Immunomodulatory oncolytic adenoviral vectors, and methods of production and use thereof for treatment of cancer

ABSTRACT

Disclosed herein are compositions and methods for treating cancer in a subject. This involves administering an oncolytic virus containing a heterologous DNA sequence encoding one or more immunomodulatory and/or immunostimulatory polypeptide(s) of interest to the subject under conditions effective to enhance an anti-tumor immune response in the subject, and to treat cancer. It also relates to a method of enhancing the delivery to and distribution within a tumor mass of therapeutic viruses.

FIELD

The invention described herein relates generally to the fields of immunology, virology, molecular biology, and more specifically to oncolytic adenoviruses having therapeutic applications.

BACKGROUND

Cancer is a leading cause of death in the United States and elsewhere. Depending on the type of cancer, it is typically treated with surgery, chemotherapy, and/or radiation. These treatments often fail, and it is clear that new therapies are necessary, to be used alone or in combination with current standards of care.

Originally conceived as solely tumor-lysing therapeutics, viruses that can preferentially target tumor cells for destruction are being used experimentally as vectors for the delivery of immune-stimulating cargo. The propagation of a lasting anti-tumor host immune response in combination with the destruction of tumor cells is described in, e.g., Lichty et al., 2014, Nature Reviews Cancer, 14: 559-567.

Clinical trials employing adenovirus, reovirus, measles, herpes simplex, Newcastle disease virus and vaccinia as oncolytic viruses have suggested that these platforms may all be safe treatment approaches.

Adenoviruses are medium-sized (90-100 nm), non-enveloped icosahedral viruses, which have double stranded linear DNA of about 36 kilobase pairs in a protein capsid. The viral capsid has fiber structures that participate in attachment of the virus to the target cell. First, the knob domain of the fiber protein binds to the receptor of the target cell (e.g., CD46 or Coxsackie and adenovirus receptor (CAR)), secondly, the virus interacts with an integrin molecule and thirdly, the virus is endocytosed into the target cell. Next, the viral genome is transported from endosomes into the nucleus and the replication machinery of the target cell is utilized also for viral purposes.

The adenoviral genome has early (E1-E4), intermediate (IX and IVa2) and late genes (L1-L5), which are transcribed in sequential order. Early gene products affect defense mechanisms, cell cycle and cellular metabolism of the host cell. Intermediate and late genes encode structural viral proteins for production of new virions.

More than 60 different serotypes of adenoviruses have been found in humans. Serotypes are classified into six subgroups A-F and different serotypes are known to be associated with different conditions i.e. respiratory diseases, conjunctivitis and gastroenteritis. Adenovirus serotype 5 (Ad-5) is known to cause respiratory diseases and it is the most common serotype studied in the field of gene therapy. In the first Ad5 vectors E1 and/or E3 regions were deleted enabling insertion of foreign DNA to the vectors.

Furthermore, deletions of other regions as well as further mutations have provided extra properties to viral vectors. Indeed, various modifications of adenoviruses have been suggested for achieving efficient anti-tumor effects.

Adenoviral vectors mediate gene transfer at a high efficacy compared to other vector systems, and they are currently the most frequently used vectors for cancer gene therapy. A non-replicating p53 expressing adenoviral vector and a replication selective virus (H101) have received regulatory approval in China. Several attempts to achieve tumor-selective control through the insertion of tumor selective promoter elements upstream of the E1 or other adenovirus critical promoters have had variable levels of success, but ultimately were limited by “leaky” gene expression of viral proteins in non-tumor cells and by reduced ability to propagate and lyse tumor cells compared to wild-type virus infections.

SUMMARY

In a first aspect, a pharmaceutical composition is provided comprising an effective amount of a recombinant adenoviral vector comprising: a transgene insertion site located between the start site of adenoviral E1b-19K and the start site of adenoviral E1b-55K, wherein a first DNA sequence and a second DNA sequence are each inserted into the transgene insertion site; wherein the first DNA sequence encodes a polypeptide selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, and a human OX40 ligand, and wherein the second DNA sequence encodes a polypeptide selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, and a human OX40 ligand; and wherein the adenoviral vector comprises a modified adenoviral E1a regulatory sequence wherein at least one Pea3 binding site, or a functional portion thereof, of the recombinant adenoviral vector is modified or deleted.

In one embodiment, the adenoviral vector comprises an IRES element or encodes a self-cleaving 2A peptide sequence between the first DNA sequence and the second DNA sequence. In another embodiment, the vector comprises a modified E3 region. In another embodiment, the vector comprises an intact E3 region. In another embodiment, the vector comprises a third DNA sequence inserted into the E3 region, wherein the third DNA sequence encodes a polypeptide selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, or a human OX40 ligand. In one embodiment, the chimeric human IL-12 polypeptide comprises a p40 polypeptide, a p35 polypeptide, and a linker polypeptide. In another embodiment, the chimeric human IL-12 polypeptide comprises a sequence as set forth in SEQ ID NO:46.

In one embodiment, the adenoviral vector comprises a nucleic acid sequence at least 95% identical in an E3 region to vector d1327. In another embodiment, the adenoviral vector comprises a nucleic acid sequence at least 85% identical in an E3 region to vector d1327. In another embodiment, the adenoviral vector comprises a nucleic acid sequence at least 75% identical in an E3 region to vector d1327.

In one embodiment, the pharmaceutical composition is formulated for systemic administration. In another embodiment, the pharmaceutical composition is formulated for intratumoral administration.

In a second aspect is provided a pharmaceutical composition comprising an effective amount of a recombinant adenoviral vector comprising: a first transgene insertion site located between the start site of adenoviral E1b-19K and the start site of adenoviral E1b-55K; a second transgene insertion site located in adenoviral E3 region; a first DNA sequence, present in the first transgene insertion site, encoding one or a plurality of polypeptides selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, and a human OX40 ligand; and a second DNA sequence, present in the second transgene insertion site, encoding one or a plurality of polypeptides selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, and a human OX40 ligand, wherein the adenoviral vector comprises a modified adenoviral E1a regulatory sequence. In one embodiment, the pharmaceutical composition further comprises a third DNA sequence inserted into a third transgene insertion site, encoding one or a plurality of polypeptides selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, and a human OX40 ligand.

In another embodiment, at least one of the first DNA sequence, the second DNA sequence, and the third DNA sequence independently comprises an IRES element and/or a self-cleaving 2A peptide. In one embodiment, at least one E1a regulatory sequence Pea3 binding site, or a functional portion thereof of the adenoviral vector, is modified or deleted. In another embodiment, a sequence between two Pea3 sites of the adenoviral vector is deleted. In one embodiment, the adenoviral vector comprises a modified E3 region. In another embodiment the adenoviral vector comprises a nucleic acid sequence at least 95% identical in an E3 region to vector d1327. In another embodiment, the adenoviral vector comprises a nucleic acid sequence at least 85% identical in an E3 region to vector d1327. In another embodiment, the adenoviral vector comprises a nucleic acid sequence at least 75% identical in an E3 region to vector d1327.

In one embodiment, the chimeric human IL-12 polypeptide comprises a p40 polypeptide, a p35 polypeptide, and a linker polypeptide.

In another aspect is provided a method for treating a tumor in a human subject in need thereof, comprising administering to the human with the tumor a therapeutic amount of the pharmaceutical composition of the first aspect by systemic or intratumoral administration.

In another aspect is provided method for treating a tumor in a human subject in need thereof, comprising administering to the human with the tumor a therapeutic amount of the pharmaceutical composition of the second aspect by systemic or intratumoral administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cartoon illustrating transcription factor binding regulation of conformational structure/activity of E1a enhancer region.

FIG. 2 is an illustration of a vector map demonstrating an adenoviral vector expressing two immunomodulatory polypeptides.

FIG. 3 is an illustration of a vector map demonstrating an adenoviral vector expressing at least one immunomodulatory polypeptide at an E4 site.

FIG. 4 is an illustration of the TRZ200 virus genome: an E1b 19k empty, E3-deleted virus.

FIG. 5 is an illustration of a vector map showing the plasmid comprising the TRZ6 hIL-12 plasmid.

FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG. 6F, FIG. 6G, FIG. 6H, FIG. 6I, FIG. 6J, FIG. 6K, and FIG. 6L are a series of graphs showing results of treatment of tumor bearing mice (n=8 in each group) with oncolytic viruses comprising various transgenes, with or without anti-PD-L1. FIG. 6A shows the activity of various oncolytic viruses compared to the empty virus (“d19k”), 38 days after cell implantation (primary tumor). Black bars represent virus alone and hatched bars represent virus+anti-PD-L1 antibody. FIG. 6B is a graph comparing the average tumor size (primary tumor) over time of tumors injected with virus buffer (black circles), empty vector (blue solid squares), anti-PD-L1 alone (antibody given on days 16, 20, 24, and 28, right-hand arrow in each pair of arrows), each of five viruses alone (CTLA-4, IL-12, IL-7, CD70, and IL-10) or combined with anti-PD-L1 antibody. FIG. 6C is a similar graph showing the remaining three viruses: OX40L, CD40L, and GM-CSF. Additional mouse treatments shown are as follows: FIG. 6D, virus buffer and control IgG only (left) and virus buffer and anti-PD-L1 antibody (right); FIG. 6E, d19k (empty virus) and control IgG only (left) and d19k and anti-PD-L1 antibody (right); FIG. 6F, CTLA-4 virus with control IgG (left) or anti-PD-L1 (right); FIG. 6G, IL-12 virus with control IgG (left) or anti-PD-L1 antibody (right); FIG. 6H, GM-CSF virus with control IgG (left) or anti-PD-L1 antibody (right); FIG. 6I, IL-7 virus with control IgG (left) or anti-PD-L1 antibody (right); FIG. 6J, CD40L virus with control IgG (left) or anti-PD-L1 antibody (right); FIG. 6K, L10 trap virus with control IgG (left) or anti-PD-L1 antibody (right); and FIG. 6L, OX40L virus with control IgG (left) or anti-PD-L1 antibody. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and the anti-PD-L1 antibody, if used (intraperitoneal, right arrows).

FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, FIG. 7F, FIG. 7G, FIG. 7H, FIG. 7I, FIG. 7J, FIG. 7K, FIG. 7L, FIG. 7M, FIG. 7N, FIG. 7O, FIG. 7P, FIG. 7Q, FIG. 7R, FIG. 7S, FIG. 7T, FIG. 7U, FIG. 7V, FIG. 7W, FIG. 7X, FIG. 7Y, FIG. 7Z, and FIG. 7AA are a series of graphs showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising various transgenes, with or without anti-PD-L1 treatment. The left-hand panel of each Figure represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). Treatments shown are as follows: FIG. 7A, virus buffer only; FIG. 7B, empty virus only (TRZ000); FIG. 7C, TRZ010 (IL-10trap)+empty virus; FIG. 7D, TRZ011 (OX40 ligand)+empty virus; FIG. 7E, TRZ009 (CD70)+empty virus; FIG. 7F, TRZ007 (IL-7)+empty virus, FIG. 7G, TRZ002 (IL-12)+empty virus; FIG. 7H, TRZ004 (GM-CSF)+empty virus; FIG. 7I, TRZ003 (flagellin)+empty virus; FIG. 7J, TRZ002+TRZ010; FIG. 7K, TRZ002+TRZ007; FIG. 7L, TRZ007+TRZ010; FIG. 7M, TRZ011+TRZ004; FIG. 7N, TRZ009+TRZ003; FIG. 7O, TRZ002+TRZ009; FIG. 7P, TRZ007+TRZ009; FIG. 7Q, TRZ007+TRZ004; FIG. 7R, TRZ002+TRZ011; FIG. 7S, TRZ010+TRZ004; FIG. 7T, TRZ002+TRZ004; FIG. 7U, virus buffer and anti-PD-L1; FIG. 7V, TRZ002+empty virus+anti-PD-L1; FIG. 7W, TRZ009+anti-PD-L1; FIG. 7X, TRZ007+empty virus+anti-PD-L1; FIG. 7Y, TRZ002+TRZ007+anti-PD-L1; FIG. 7Z, TRZ007+TRZ009+anti-PD-L1; and FIG. 7AA, TRZ002+TRZ009+anti-PD-L1, 7BB.

FIG. 8A and FIG. 8B are schematic representations of the rationale for modifications of the E1a Enhancer region of adenoviral vectors, in which Pea3 sites are altered, moved, or deleted in order to produce vectors with a variety of expression characteristics. FIG. 8A is an illustration of a wild-type adenoviral genome (top) and the TAV-255 adenoviral construct in which residues −305 to −255 are deleted (bottom). FIG. 8B is an illustration of the potential method of action of the TAV-255 construct, in which the deletion removes Pea3 sites II and III, which moves Pea3 sites IV and V closer to the promoter as distal control elements.

FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E, FIG. 9F, FIG. 9G, FIG. 9H, FIG. 9I, FIG. 9J, FIG. 9K, FIG. 9L, FIG. 9M, FIG. 9N, FIG. 9O, FIG. 9P, FIG. 9Q, FIG. 9R, FIG. 9S, FIG. 9T, FIG. 9U, FIG. 9V, FIG. 9W, FIG. 9X, FIG. 9Y, and FIG. 9Z are a series of graphs showing treatment of tumor bearing mice (n=8 in each group) with dual transgene (either expressed from both E1 and E3, or dual transgenes expressed from either E1 or E3) oncolytic viruses comprising various transgenes, with either anti-PD-L1 antibody or anti-PD-1 antibody treatment. FIG. 9A is a cartoon of the contrast between the single transgene constructs (top) used in the virus mixing examples, and the dual transgene vectors used to make the E1/E3 transgene adenoviruses. FIG. 9B shows the results of mice injected i.t. with Empty virus +/−anti-PD-L1 or with TRZ-409 (IL-12/IL-7 dual transgene) +/−anti-PD-L1. The tumor volume of the injected tumor is shown in the left panel, and the tumor volume of the contralateral tumor is shown in the right panel. For each pair of arrows, the left arrow indicates virus injection and the right arrow indicates anti-PD-L1 injection. As can be seen for both the primary and contralateral tumors, TRZ-409 injection reduced tumor volume significantly compared to empty virus. The combination with anti-PD-L1 was slightly more efficacious in this study. FIG. 9C shows the results of a second experiment using the inverse of TRZ-409, TRZ-403, in which the IL-7 transgene occupies the E1 region and the IL-12 gene occupies the E3 region (which has a stronger promoter than the E1). As can be seen in the Figure, TRZ-403 strongly inhibits primary and distant tumor growth, with or without anti-PD-L1. Plasma levels of IL-12 and IFN-γ (FIG. 9D) for all mice injected with TRZ-403 show that expression of the transgenes is well tolerated. The study was extended for all mice having tumors over 500 mm³. As can be seen in the FIG. 9E, mice injected with TRZ-403 had a much higher percentage of survival over TRZ-409 at the time end of the study (70 days). Mice having primary tumors injected with TRZ-403 (IL-7+IL-12), TRZ-403+anti-PD-L1, TRZ-403+control IgG, or TRZ-409 (IL-12+IL-7), and controls including untreated mice and mice injected with empty vector TRZ-d19K with anti-PD-L1, anti-PD-1, or a control IgG. As can be seen in the Figure, mice injected with TRZ-403 had a much higher percentage of survival over TRZ-409 at the time end of the study (70 days). Both TRZ-403 and TRZ-409 showed efficacy over the controls; all mice receiving control injections were deceased by day 56 of the study. FIG. 9F compares a mixture of two single transgene viruses with a dual transgene virus having the same transgenes. As can be seen in the primary tumor (left panel) TRZ-403 showed the most efficacy in reducing tumor volume, followed by the mixture of IL-7 and IL-12 single transgene viruses. The single transgene IL-12 virus showed a small amount of efficacy by comparison. In the contralateral tumor, however (right panel) only the dual transgene virus, TRZ-403, reduced the tumor volume, showing significant superiority over the mixture of viruses. FIG. 9G, FIG. 9H, FIG. 9I, FIG. 9J, FIG. 9K, FIG. 9L, FIG. 9M, FIG. 9N, FIG. 9O, FIG. 9P, FIG. 9Q, FIG. 9R, FIG. 9S, FIG. 9T, FIG. 9U, FIG. 9V, FIG. 9W, FIG. 9X, FIG. 9Y, and FIG. 9Z show comparisons of anti-tumor activity of various dual transgene viruses and controls, including comparison with antibody treatments. FIG. 9G, FIG. 9H, FIG. 9I, FIG. 9J, FIG. 9K, FIG. 9L, FIG. 9M, FIG. 9N, FIG. 9O, FIG. 9P, FIG. 9Q, FIG. 9R, FIG. 9S, FIG. 9T, FIG. 9U, FIG. 9V, FIG. 9W, FIG. 9X, FIG. 9Y, and FIG. 9Z show the primary tumor in the left panel and the contralateral tumor in the right panel. All viruses were administered intratumorally and the antibodies were administered intraperitoneally. Viral dose regimens vary and are tabulated in Table 7 (Example 12); all antibodies are administered at a dose of 2504. FIG. 9G shows the tumor activity in untreated animal controls; FIG. 9H shows TRZ000 (E1-Empty/E3 Intact); FIG. 9I, FIG. 9J, and FIG. 9K show TRZ002 (E1-mIL-12/E3 Del); FIG. 9L, FIG. 9M, and FIG. 9N show TRZ510 (E1-mIL-7-P2A-mIL-12/E3 Del); FIG. 9O, FIG. 9P, and FIG. 9Q show TRZ021 (E1-empty/E3-mIL-2); FIG. 9R, FIG. 9S, FIG. 9T, FIG. 9U, and FIG. 9V show TRZ421 (E1-mIL-12/E3-mIL-2); FIG. 9W shows the combination of TRZ002 (E1-mIL-12/E3 Del)+Anti-CTLA4 antibody (see Table 7 for doses); FIG. 9X shows anti-PD-1 antibody treatment alone, FIG. 9Y shows anti-CTLA4 antibody alone, and FIG. 9Z shows results of treatment with the combination of anti-CTLA-4 and anti-PD-1 antibodies (both from BioXCell).

FIG. 10A FIG. 10B, FIG. 10C, FIG. 10D, FIG. 10E, FIG. 10F, and FIG. 10G are a series of graphs showing measurement of in vitro expression of transgenes cloned into the E1 or E3 site of oncolytic adenoviruses. A549 or ADS12 cells were infected with TRZ000 (empty virus), TRZ403 (E1 IL-7/E3 IL-12), TRZ416 (E1 OX40L/E3 IL-12), TRZ421 (E1 IL-12/E3 IL-2), TRZ510 (E1 IL-7-P2A-IL-12/E3), or TRZ512 (E1-IL-12-P2A-IL-7/E3) at a multiplicity of infection (MOI) of 10 or 25. Forty-eight hours after infection, the supernatants were harvested, clarified by centrifugation, and assayed for protein expression by standard ELISA methods (mouse IL-12, mouse IL-2, and mouse IL-7) using Quantikine® kits, (R&D Systems); and mouse OX40L using a DuoSet® kit, (R&D Systems). Expression of transgenes from the adenovirus E1 site is shown for IL-7 (TRZ403, FIG. 10A, A549 cells), IL-7 (TRZ510 and TRZ512, FIG. 10B, ADS-12 cells), OX40L (TRZ416, FIG. 10C, A549 cells), IL-12 (TRZ421), FIG. 10D, A549 cells), IL-12 (TRZ510 and TRZ512, FIG. 10E, ADS-12 cells). Expression of transgenes from the E3 site is shown for IL-12 (TRZ403 and TRZ416, FIG. 10F, A549 cells) and IL-2 (TRZ421, FIG. 10G, A549 cells). As can be seen in the Figures, all transgenes were expressed well from either the E1 or E3 site.

FIG. 11A FIG. 11B, FIG. 11C, FIG. 11D, and FIG. 11E are a series of graphs showing measurement of in vitro function of transgenes cloned into the E1 or E3 site of oncolytic adenoviruses. A549 cells were infected with TRZ510 (E1 IL-7-P2A-IL-12/E3), TRZ512 (E1 IL-12-P2A-IL-7/E3), TRZ403 (E1 IL-7/E3 IL-12), and TRZ416 (E1 OX40L/E3 IL-12) at a multiplicity of infection (MOI) of 10 or 25. Forty-eight hours after infection, the supernatants were harvested, clarified by centrifugation, and assayed for biological function of secreted IL-12 using HEK-Blue™ IL-12 cells or of secreted IL-2 using CTLL-2 cells. Function of IL-12 transgenes in HEK-Blue IL-12 cells is shown for IL-12 in supernatants expressed from the E1 region (FIG. 11A, TRZ510; FIG. 11B, TRZ512) and from the E3 region (FIG. 11C, TRZ403; FIG. 11D, TRZ416). Function of IL-2 transgenes in CTLL-2 cells is shown for supernatants expressed from the E3 region is shown in FIG. 11E (TRZ421). As can be seen in the Figures, all transgenes were functional when expressed from the E1 or E3 sites, including those connected with a P2A sequence.

DEFINITIONS

The term “replicating virus” is meant to include a virus that undergoes the process of intracellular viral multiplication, consisting of the synthesis of proteins, nucleic acids, and sometimes lipids, and their assembly into a new infectious particle.

As used herein, the term “adenovirus” refers to any of a group of DNA-containing viruses (small infectious agents) that cause conjunctivitis and upper respiratory tract infections in humans. Adenoviral vectors are described in Peng, Z., “Current Status of Gendicine in China: Recombinant Human Ad-p53 Agent for Treatment of Cancers,” Hum Gene Ther 16:1016-1027 (2005); No authors listed, “The End of the Beginning: Oncolytic Virotherapy Achieves Clinical Proof-of-concept,” Mol Ther 13:237-238 (2006); Vile et al., “The Oncolytic Virotherapy Treatment Platform for Cancer: Unique Biological and Biosafety Points to Consider,” Cancer Gene Ther 9:1062-1067 (2002); Harrison et al., “Wild-type Adenovirus Decreases Tumor Xenograft Growth, but Despite Viral Persistence Complete Tumor Responses are Rarely Achieved—Deletion of the Viral E1b-19-kD Gene Increases the Viral Oncolytic Effect,” Hum Gene Ther 12:1323-1332 (2001); Kim et al., “Clinical Research Results with d11520 (Onyx-015), a Replication-selective Adenovirus for the Treatment of Cancer: What Have We Learned?,” Gene Ther 8:89-98 (2001); and Thorne et al., “Oncolytic Virotherapy: Approaches to Tumor Targeting and Enhancing Antitumor Effects,” Semin Oncol 32:537-548 (2005), each of which is hereby incorporated by reference in their entirety. Adenoviral positions referenced herein are to positions in Adenovirus type 5 (GenBank 10 accession #M73260; the virus is available from the American Type Culture Collection, Rockville, Md., U.S.A., under accession number VR-5). It will be understood that corresponding positions can be identified in other adenovirus vectors by alignment using BLAST 2.0 under default settings (Altschul et al., J. Mol. Biol. 215:403-410 (1990)). Software for performing BLAST analyses is publicly available on the Web through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).

Current research in the field of viral vectors is producing improved viral vectors with high-titer and high-efficiency of transduction in mammalian cells (see, e.g., U.S. Pat. No. 6,218,187 to Finer et al., which is hereby incorporated by reference in its entirety). Such vectors are suitable in the present invention, as is any viral vector that comprises a combination of desirable elements derived from one or more of the viral vectors described herein. It is not intended that the expression vector be limited to a particular viral vector.

Certain “control elements” or “regulatory sequences” can also be incorporated into the vector-construct. The term “control elements” refers collectively to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, and the like, which collectively provide for the replication, transcription, and translation of a coding sequence(s) in a recipient cell. Not all of these control elements need always be present so long as the selected coding sequence is capable of being replicated, transcribed, and translated in an appropriate host cell.

The term “promoter region” is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3′-direction) coding sequence. Transcriptional control signals in eukaryotes comprise “promoter” and “enhancer” elements. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources, including genes in yeast, insect, and mammalian cells, and viruses. Analogous control elements, i.e., promoters, are also found in prokaryotes. Such elements may vary in their strength and specificity. For example, promoters may be “constitutive” or “inducible.”

A constitutive promoter is a promoter that directs expression of a gene throughout the development and life of an organism. Examples of some constitutive promoters that are widely used for inducing expression of transgenes include the cytomegalovirus (CMV) early promoter, those derived from any of the several actin genes, which are known to be expressed in most cells types (U.S. Pat. No. 6,002,068 to Privalle et al., which is hereby incorporated by reference in its entirety), and the ubiquitin promoter, which is a gene product known to accumulate in many cell types.

To ensure efficient expression, 3′ polyadenylation regions can be present to provide for proper maturation of the mRNA transcripts. The 3′ polyadenylation region will preferably be from the adenovirus sequence downstream of the inserted transgene, but the native 3′-untranslated region of the immunomodulatory gene may be used, or an alternative polyadenylation signal from, for example, SV40, particularly including a splice site, which provides for more efficient expression, could also be used. Alternatively, the 3′-untranslated region derived from a gene highly expressed in a particular cell type could be fused with the immunomodulatory gene.

“Inhibitors,” “activators,” and “modulators” of expression or of activity are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo assays for expression or activity of a described target protein, e.g., ligands, agonists, antagonists, and their homologs and mimetics. The term “modulator” includes inhibitors and activators. Inhibitors are agents that, e.g., inhibit expression or bind to, partially or totally block stimulation or protease inhibitor activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of the described target protein, e.g., antagonists. Activators are agents that, e.g., induce or activate the expression of a described target protein or bind to, stimulate, increase, open, activate, facilitate, enhance activation or protease inhibitor activity, sensitize or up regulate the activity of described target protein (or encoding polynucleotide), e.g., agonists. Modulators include naturally occurring and synthetic ligands, antagonists and agonists (e.g., small chemical molecules, antibodies and the like that function as either agonists or antagonists). Such assays for inhibitors and activators include, e.g., applying putative modulator compounds to cells expressing the described target protein and then determining the functional effects on the described target protein activity, as described above. Samples or assays comprising described target protein that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of effect. Control samples (untreated with modulators) are assigned a relative activity value of 100%. Inhibition of a described target protein is achieved when the activity value relative to the control is about 80%, optionally 50% or 25, 10%, 5% or 1%. Activation of the described target protein is achieved when the activity value relative to the control is 110%, optionally 150%, optionally 200, 300%, 400%, 500%, or 1000-3000% or higher.

“Antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

Naturally occurring immunoglobulins have a common core structure in which two identical light chains (about 24 kD) and two identical heavy chains (about 55 or 70 kD) form a tetramer. The amino-terminal portion of each chain is known as the variable (V) region and can be distinguished from the more conserved constant (C) regions of the remainder of each chain. Within the variable region of the light chain is a C-terminal portion known as the J region. Within the variable region of the heavy chain, there is a D region in addition to the J region. Most of the amino acid sequence variation in immunoglobulins is confined to three separate locations in the V regions known as hypervariable regions or complementarity determining regions (CDRs) which are directly involved in antigen binding. Proceeding from the amino-terminus, these regions are designated CDR1, CDR2 and CDR3, respectively. The CDRs are held in place by more conserved framework regions (FRs). Proceeding from the amino-terminus, these regions are designated FR1, FR2, FR3, and FR4, respectively. The locations of CDR and FR regions and a numbering system have been defined by, e.g., Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, U.S. Government Printing Office (1991)).

An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V_(L)) and variable heavy chain (V_(H)) refer to these light and heavy chains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′₂, a dimer of Fab which itself is a light chain joined to V_(H)-C_(H1) by a disulfide bond. The F(ab)′₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)′₂ dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab with part of the hinge region (see FUNDAMENTAL IMMUNOLOGY (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).

For preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)). “Monoclonal” antibodies refer to antibodies derived from a single clone. Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)).

A “chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

A “humanized” antibody is an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts. See, e.g., Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988); Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3):169-217 (1994).

As used herein, the terms “treat” and “treating” in the context of the administration of a therapy refers to a treatment/therapy from which a subject receives a beneficial effect, such as the reduction, decrease, attenuation, diminishment, stabilization, remission, suppression, inhibition or arrest of the development or progression of cancer, or a symptom thereof. In certain embodiments, the treatment/therapy that a subject receives results in at least one or more of the following effects: (i) the reduction or amelioration of the severity of cancer and/or a symptom associated therewith; (ii) the reduction in the duration of a symptom associated with cancer; (iii) the prevention in the recurrence of a symptom associated with cancer; (iv) the regression of cancer and/or a symptom associated therewith; (v) the reduction in hospitalization of a subject; (vi) the reduction in hospitalization length; (vii) the increase in the survival of a subject; (viii) the inhibition of the progression of cancer and/or a symptom associated therewith; (ix) the enhancement or improvement the therapeutic effect of another therapy; (x) a reduction or elimination in the cancer cell population; (xi) a reduction in the growth of a tumor or neoplasm; (xii) a decrease in tumor size; (xiii) a reduction in the formation of a tumor; (xiv) eradication, removal, or control of primary, regional and/or metastatic cancer; (xv) a decrease in the number or size of metastases; (xvi) a reduction in mortality; (xvii) an increase in cancer-free survival rate of patients; (xviii) an increase in relapse-free survival; (xix) an increase in the number of patients in remission; (xx) a decrease in hospitalization rate; (xxi) the size of the tumor is maintained and does not increase in size or increases the size of the tumor by less 5% or 10% after administration of a therapy as measured by conventional methods available to one of skill in the art, such as MRI, X-ray, and CAT Scan; (xxii) the prevention of the development or onset of cancer and/or a symptom associated therewith; (xxiii) an increase in the length of remission in patients; (xxiv) the reduction in the number of symptoms associated with cancer; (xxv) an increase in symptom-free survival of cancer patients; and/or (xxvi) limitation of or reduction in metastasis. In some embodiments, the treatment/therapy that a subject receives does not cure cancer, but prevents the progression or worsening of the disease. In certain embodiments, the treatment/therapy that a subject receives does not prevent the onset/development of cancer, but may prevent the onset of cancer symptoms.

As used herein, the term “in combination” in the context of the administration of (a) therapy(ies) to a subject, refers to the use of more than one therapy. The use of the term “in combination” does not restrict the order in which therapies are administered to a subject. A first therapy can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy to a subject.

The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region, e.g., of the entire polypeptide sequences or specific region, if indicated), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.”

For sequence comparison, one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of, e.g., a full-length sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison are conducted by a BLAST 2.0 algorithm, which is described in Altschul et al. (1990) J. Mol. Biol. 215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

DETAILED DESCRIPTION

Disclosed herein are compositions and methods of treating cancer in a subject. Provided are replicating viral vectors, specifically oncolytic adenoviral vectors, that contain one or more recombinant nucleic acid sequences encoding therapeutic polypeptides.

Oncolytic adenovirus (AV) expressing a transgene display highly oncolytic and immunogenic properties. Therefore, the recombinant adenoviral vectors provided herein have the potential to have broad activity against a primary tumor infected with adenovirus but also against tumors in the metastatic disease setting. In some embodiments, the metastatic tumors need not be directly injected with virus nor does the virus injected into the primary site travel to the metastatic setting (e.g. through the blood stream).

However, due to expression of an immune stimulatory transgene from the adenovirus (for example, including but not limited to an E1b 19K deleted region in an exemplary vector such as TAV-255 (e.g., TAV-255 Δ19)), the immune system will be primed to fight cancer systemically in the entire body of the cancer patient as long as the metastatic tumor cells express the same tumor antigens as the primary tumor cells. Since metastases are derived from the primary tumors and genetically very similar to the primary tumor cells, metastatic tumor growth will be inhibited, in some embodiments, to the same extent as the primary tumor.

Provided are adenoviruses as described herein. In another aspect, a cell transformed with any one of the recombinant adenoviruses described herein is provided.

In another aspect, a method is provided of selectively expressing a peptide in a target cell comprises contacting the target cell with any one of the recombinant adenoviruses described herein. In one aspect, the recombinant adenovirus comprises a E1a regulatory sequence deletion mutant operably linked to a nucleotide sequence encoding a peptide, e.g., a peptide associated with viral replication or with cancer.

Also provided herein are methods of adenoviral therapy that utilize the oncolytic adenoviruses of the instant invention as adenoviral vectors that express one, two, or more recombinant immunomodulatory genes. The oncolytic adenovirus contains a heterologous gene that encodes a therapeutic protein, incorporated within the viral genome, such that the heterologous gene is expressed within an infected cell. A therapeutic protein, as used herein, refers to a protein that provides one or more therapeutic benefit when expressed in a given cell. In particular, the therapeutic benefit includes recruitment of the host immune system to the tumor.

Modified Regulatory Regions

E1a is the first protein produced by an adenovirus upon infection of a cell, activating other adenoviral promoters and facilitating infected cells to enter cell division. Rendering expression of this protein under tumor-selective control is an effective means of limiting expression of viral proteins and oncolysis to tumor cells.

Normal cells require mitogenic growth signals (GS) before they can move from a quiescent state into an active proliferative state. Tumor cells are able to generate many of their own growth signals or mimic normal growth signals, and transcription factors such as E2F1 and Pea3 are commonly overexpressed in tumor cells at levels that can cooperate in forming conformational structures optimal for driving E1a transcription during adenovirus infection and replication. (Hanahan D, Weinberg R A. The Hallmarks of Cancer. Cell 2000; 100: 57-70; de Launoit Y, Chotteau-Lelievre A, Beaudoin C, Coutte L, Netzer S, Brenner C et al., The PEA3 Group of ETS-Related Transcription Factors: Role in Breast Cancer Metastasis, Adv Exp Med Biol 2000; 480: 107-116; Bruder J T, Hearing P: Cooperative Binding of EF-1A to the E1A Enhancer Region Mediates Synergistic Effects on E1A Transcription During Adenovirus Infection. J Virol 1991; 65:5084-5087).

Small deletions selectively targeting the binding sites for E2F1 and Pea3 sites in the E1a enhancer region are an alternative and less disruptive method than complete replacement of the E1a enhancer region with a transcriptionally restricted promoter element. Cooperative binding and transcriptionally optimized conformation of the E1a enhancer region could still take place due to the over-abundance of transcription factors found in tumor cells, while in normal, non-dividing cells, the disruption of binding sites would further inhibit the ability to form optimized conformations in the limiting level of mitogenic growth signals.

Thus, disclosed herein are methods of engineering adenoviral vectors via the Pea3 binding sites. The five Pea3 transcription factor binding sites, also known as E1AF (or originally as EF-1A-enhancer binding factor to the E1a core motif) have differential effects on the production of E1a mRNA levels as demonstrated by specific deletions of individual and paired sites. The murine Pea3 sites are described herein as Pea3 sites I, II, III, IV, and V. The main binding sites for Pea3 are sites I and III, while sites II, IV, and V are slightly degenerate versions. Pea3 binds cooperatively between sites II and III, IV and V, and II and I, and this cooperative binding activates E1a transcription. (see, e.g., Hearing (J. Virol 65, 1991, Mol Cell Biol 9, 1989, and Nuc Acids Res 20, 1992). Pea3 itself is a dimer of both a and β subunits, where the α subunit makes the primary DNA contact and the β subunit forms a heteromultimeric complex with the β subunit both in solution or on a dimeric binding site. During normal infection conditions, binding at sites I and III, followed by the cooperative binding at site II would cause conformational changes that serve to bring this protein/DNA complex closer to the activation transcription factor (ATF) binding site and TATA box for full activation of E1a mRNA expression. The two lower affinity sites, Pea3 IV and Pea3 5, do not appear to contribute much to activation under these normal circumstances, as they may be too far away or unoccupied. Deletion of site II causes the greatest reduction in transcriptional activity, pointing to the importance of the cooperative binding effect it has for sites III and I in activating transcription, presumably through a conformational change. Deletion of either site III or I had much less of a reduction, since presumably you could still have cooperative binding between site II and the remaining site III or I. Deletion of both sites I and III (but not II) reduces transcription to the levels seen with Pea3 site II deletion alone, and the combination of deleting both sites III and II, or sites II and I also results in similar (but not greater) levels of reduction.

Deletion of the region encompassing sites III and II removes this cooperative binding capacity derived from site II, but it also moves sites IV and V closer to site I. Although lower affinity, sites IV and V do show cooperative binding, and by moving them closer to site I, binding at these three sites may be able to mimic the conformation change normally occurring with binding of sites I-III, leading to transcriptional activation. Because sites IV and V are lower affinity sites, there may not be enough of this transcription factor around in normal cells to get full binding on the deleted construct for activation, but there are several publications reporting increased levels of E1AF/Pea3 with tumor progression and invasiveness (i.e., Horiuchi S. et. al., J Pathol 2003 August; 200(5): 568-76), indicating that tumor cells may have enough E1AF to bind the lower affinity binding sites IV and V, along with site I, and potentially lead to a conformational change needed to activate transcription of E1a in tumor but not normal cells with this deleted virus.

The present invention provides replicating adenoviruses. In some embodiments, the replicating vectors of the instant invention contain recombinant (e.g., exogenous) transgene(s) expressing immunomodulatory polypeptides that are controlled by endogenous adenovirus early promoters, thereby driving meaningfully higher expression levels than can be generally achieved in replication deficient viruses. In addition to the enhanced anti-tumor efficacy resulting from tumor-specific oncolysis from the replicating adenovirus, the higher expression levels from the recombinant transgenes in a replicating virus results in enhanced immunomodulatory effect(s) over the lower expression levels in replication deficient viruses.

In one aspect, a recombinant virus comprises a modified E1a regulatory sequence, wherein at least one Pea3 binding site, or a functional portion thereof, is deleted. In one aspect, a sufficient number of nucleotides in the range of −305 to −141 are retained to maintain functional activity of the Ad packaging signal function and (near) optimal transcription of the E1a protein in tumor but not growth arrested normal cells.

Additional deletions or modifications of individual or combinations of Pea3V, Pea3IV, Pea3III, Pea3II and/or E2F1 binding sites between base pairs −394 and −218 designed to inhibit binding of Pea3 and/or E2F1 to these sites and take part in cooperative binding induced conformational changes in the E1a enhancer region are also encompassed by this description.

In one aspect, at least one of Pea3 II, Pea3 III, Pea3 IV, and Pea3 V, or a functional portion thereof, is deleted or modified (e.g., at least one nucleotide of the sequence is changed or an additional nucleotide is inserted into the sequence). In another aspect, at least one of Pea3 II and Pea3 III, or a functional portion thereof, is deleted or modified. In one aspect, Pea3 II or a functional portion thereof, and Pea3 III or a functional portion thereof, is deleted or modified. In another aspect, at least one of Pea3 IV and Pea3 V, or a functional portion thereof, is deleted or modified. In another aspect, Pea3 I, or a functional portion thereof, is retained. By “retained” is meant that the element is present in the recombinant adenoviral vector, preferably at the same location as a reference adenoviral vector. In one aspect, at least one E2F1 binding site, or a functional portion thereof, is retained.

In one aspect, the vector is d1309-6, TAV-255, d155, d1200, d1230, or d1200+230. In another aspect, the vector is TAV-255. In another aspect, the E1a deletions in d1309-6, TAV-255, d155, d1200, d1230, d1200+230, or other E1a modifications affecting Pea3 and/or E2F1 binding sites between −394 to −218, are paired with a non-d1309 based E3 deletion such as the E3 deletion found in pBHG10 (Microbix, Ad5 base pairs (bp) 28133-30818), d1327 (Ad5 bp 28593-30470) or a similar size E3 deletion such that >3 kb of exogenous DNA can be successfully packaged and expressed from a recombinant adenovirus with the E1a deletions listed above, in combination with a deletion between the start site of the E1b 19K protein and the start site of the E1b 55K protein of approximately 202 base pairs. In one aspect, the vector is a d1309 vector having one or more mutations in reference to the wild type sequence of Ad5 (see, e.g., Chroboczek et al., Virology (1992) January; 186(1):280-5, herein incorporated by reference), including a disruption in the coding sequences for one or more of the 10.4K, 14.5K, and 14.7K proteins in the E3 region.

In one aspect, a recombinant virus selectively expresses at least one E1a isoform, e.g., Ela-12S or Ela-13S. In one aspect, the sequence encoding the E1a isoform is operably linked to a modified E1a regulatory sequence, wherein at least one Pea3 binding site, or a functional portion thereof, is deleted or modified.

In one aspect, a recombinant virus comprises a DNA sequence, e.g., a transgene, inserted into an E1b-19K insertion site. In one aspect, the insertion site is located between the start site of E1b-19K and the start site of E1b 55K. In another aspect, the insertion site comprises a deletion of 202 base pairs following the start site of E1b-19K. A transgene (also, “insert”) may be a full natural sequence of the gene of interest or a fragment thereof. It may be modified too include a Kozak sequence, stop codon, or other regulatory elements. A transgene may include one or more endonuclease restriction sites.

In another aspect, expression the transgene is operably linked to a modified E1a regulatory sequence, wherein at least one Pea3 binding site, or a functional portion thereof, is modified or deleted. The transgene may be located in the E1, E2, E3 and/or E4 regions of the adenovirus but its expression is controlled by E1a mediated activation of the endogenous upstream adenovirus promoter, generating high levels of transgene expression only during E1a mediated viral replication.

In specific embodiments, the transgene is located in the E3 region. Exogenous transgenes can be inserted into one or more deleted regions of the adenovirus E3 region, generally such transgenes are inserted into the adenoviral vector as to have their expression controlled by the endogenous E3 promoter (Luo J et al, Clin Cancer Res 2008: 14 2450-2457). This results in high levels of transgene expression that are specifically expressed during periods of viral replication, as the E3 promoter only becomes transcriptionally active during these times. Using a modified adenovirus backbone, such as the E1a enhancer modification described herein, to limit viral replication to infected tumor cells, expression from the introduced transgene is also limited to infected tumor cells and, generally, not normal cells. In certain embodiments, the entire E3 region, or a substantial portion of the E3 region, is deleted.

In specific embodiments, the transgene is located in the E4 region. In a similar fashion to inserting exogenous transgenes into deleted regions in the E3 or E1 regions of adenovirus, there are regions in the E4 region of adenovirus that can be deleted without significant effect on viral growth characteristics (Gao, G P et al, J. Virol 1996: 70; 8934-8943). Generally, at least ORF3 and/or ORF6 are retained in the adenoviral vector in which the remainder of the E4 region. It should be possible to insert a foreign transgene into one of these deletions in the E4 region and drive expression of this gene with the endogenous E4 promoter of adenovirus, restricting high level expression of this gene to conditions where viral replication is expected to occur.

Tumor-Directed Recombinant Immunomodulatory Polypeptide Production.

The adenoviruses described herein can be engineered to express an immunomodulatory agent or immunomodulatory polypeptide, e.g., a polypeptide agonist of a co-stimulatory signal of an immune cell. In some embodiments, the polypeptide agonist is an agonist of a T effector cell and/or the polypeptide agonist functions as a polypeptide antagonist of an inhibitory signal of an immune cell such as a regulatory T cell. As provided herein, an “immunomodulatory protein” or an “immunomodulatory polypeptide” includes any polypeptide or set of polypeptides capable of modulating (e.g., stimulating) the anti-tumor immune response induced by the adenovirus. Generally, an “immunomodulatory polypeptide” includes a desired immunostimulatory activity. An immunomodulatory polypeptide can include a set of polypeptides, linked or unlinked, that can form a multimer (e.g., a dimer) capable of modulating the anti-tumor immune response induced by adenovirus, e.g., IL-12 dimer formed from p40 and p35, with or without a linker. Immunomodulatory polypeptides can be full length proteins as occur in nature or can be fusions, variants, or fragments thereof that retain at least about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the immunomodulatory activity of the full-length protein.

As used herein, the term “agonist(s)” refers to a molecule(s) that binds to another molecule and induces an increased biological reaction. In a specific embodiment, an agonist is a molecule that binds to a receptor on a cell and triggers or stimulates one or more signal transduction pathways. For example, an agonist can include an antibody or ligand that binds to a receptor on a cell and induces one or more signal transduction pathways. In other embodiments, the agonist facilitates the interaction of the native ligand with the native receptor. As used herein, the term “antagonist(s)” refers to a molecule(s) that inhibits the action of another molecule, optionally without provoking an independent biological response itself. In a specific embodiment, an antagonist is a molecule that binds to a receptor on a cell and blocks or dampens the biological activity of an agonist. For example, an antagonist can include an antibody or ligand that binds to a receptor on a cell and blocks or dampens binding of the native ligand to the cell, optionally without inducing one or more signal transduction pathways. Another example of an antagonist includes an antibody or soluble receptor that competes with the native receptor on cells for binding to the native ligand, and thus, blocks or dampens one or more signal transduction pathways induced when the native receptor binds to the native ligand. A further example of an antagonist includes an antibody or soluble receptor that competes with the native receptor on cells for binding to the native ligand or blocks receptor internalization, and thus, blocks or dampens one or more signal transduction pathways induced when the native receptor binds to the native ligand.

In specific embodiments, the immunomodulatory polypeptide expressed by the adenovirus is a costimulatory ligand, e.g., GITR ligand (GITRL), OX40 ligand (OX40L), or CD40 ligand (CD40L). Expression of one or more costimulatory ligands in the tumor microenvironment and the specific binding to the cognate receptor results in an increase in activity of a tumor-infiltrating lymphocyte (TIL), such activity including TIL proliferation and cytokine release, thereby increasing the anti-tumor activity of the pharmaceutical composition.

In specific embodiments, the immunomodulatory polypeptide expressed by the adenovirus is a pro-inflammatory cytokine, e.g., GMCSF, IL-7, IL-12, or an IL-15 hybrid (e.g., a hybrid of IL-15 and IL-15 receptor alpha). Expression of one or more pro-inflammatory cytokine in the tumor microenvironment results in a tumor-localized increase in the inflammatory milieu, thereby increasing the anti-tumor activity of the pharmaceutical composition, and also increasing safety of the composition by decreasing or eliminating undesired effects of systemic administration of a pro-inflammatory cytokine.

In specific embodiments, the immunomodulatory polypeptide expressed by the adenovirus is an inhibitor (e.g., a “receptor trap” or a “trap”) of an inhibitory cytokine, e.g., IL-10 or IL-27. In other embodiments, the immunomodulatory agent/inhibitor of an inhibitory cytokine is an antibody against, e.g., TGFB or IL-10R. Such inhibitory cytokines decrease T effector cell function. Expression of one or more inhibitory cytokine receptor traps in the tumor microenvironment results in a tumor-localized binding to and neutralization of the inhibitory cytokine, thereby reducing or preventing its inhibitory activity and increasing the anti-tumor activity of the pharmaceutical composition, and also increasing safety of the composition by decreasing or eliminating undesired effects of systemic administration of a blockage of an inhibitory cytokine.

In specific embodiments, the immunomodulatory polypeptide expressed by the adenovirus is an initiator of a localized immune response, e.g., a protein (e.g., secreted flagellin) that activates a toll-like receptor ligand such as TLR-5. Expression of one or more immune response initiators in the tumor microenvironment results in a tumor-localized immune response (e.g., infiltration of TILs and antigen presenting cells (APCs) and increasing the anti-tumor activity of the pharmaceutical composition.

In specific embodiments, the immunomodulatory polypeptide expressed by the adenovirus is an inhibitor (e.g., an antibody antagonist) of a co-inhibitory checkpoint molecule, e.g., CTLA4. Such inhibitory checkpoint molecules decrease T effector cell function. Expression of one or more inhibitory checkpoint molecules at the surface of an activated T cell (e.g., an activated T effector cell) attenuates the functional activity of the T cell. Thus, expression of an antagonist of the co-inhibitory checkpoint molecule in the tumor microenvironment results in blocking the co-inhibitory activity and increasing the anti-tumor activity of the pharmaceutical composition, and also increasing safety of the composition by decreasing or eliminating undesired effects of systemic administration of a blockage of a co-inhibitory checkpoint molecule.

In specific embodiments, the immunomodulatory polypeptide expressed by the adenovirus is a cluster of differentiation (CD) molecule or a ligand of a cluster of differentiation (CD) molecule such as CD27, e.g., CD70. Expression of a CD27 ligand in the tumor microenvironment results in a tumor-localized NK-mediated tumor clearance and promotes the adaptive immune response against the tumor, thereby increasing the anti-tumor activity of the pharmaceutical composition. Other suitable CD molecules include CD1d, CD2, CD3, CD4, CD5, CD6, CD7, CD8a, CD8b, CD9, CD21, CD25, CD37, CD40, CD49b, CD53, CD57, CD69, CD80, CD81, CD82, CD86, CD99, CD103, CD134, CD152, CD154, CD165, CD244, CD267, CD272, CD273, CD274, CD278, CD305, CD314, CD357, and CD360, or the ligands thereof, or any modulator (e.g., a stimulator or an inhibitor) thereof.

In specific embodiments, the immunomodulatory polypeptide expressed by the adenovirus is selected from a polypeptide provided in Table 1.

TABLE 1 Exemplary Immunomodulatory polypeptides SEQ ID Accession NO: Immunomodulatory Polypeptide Number(s) 1 GITR-Ligand (GITR-L; TNFSF18) NP_005083 2 GITR-Ligand (GITR-L; TNFSF18) NM_183391 3 CD28 ligand or agonist (CD80) EAW79565 4 TNFα CAA78745 5 Non-cleavable TNFα NM_013693 6 GM-CSF NM_009969 7 ICOS ligand or agonist NP_056074 8 4-1BB ligand or agonist AAA53134 (human) 9 OX40 ligand or agonist CAE11757 10 OX40 ligand NM_009452 11 CD40 ligand or agonist NP_000065 12 CD40 ligand NM_011616 13 CD27 ligand or agonist AAA36175 14 CD70 ligand or agonist NM_011617 15 Interleukin-2 (IL-2) AAB46883 16 Interleukin-7 (IL-7) AAH47698 17 Interleukin-7 (IL-7) NM_00837 18 Interleukin-12 (IL-12) alpha subunit AAD16432 19 Interleukin-12 (IL-12) beta subunit NP_005526 20 Interleukin-12 fusion polypeptide N/A 21 IL-15 CAA62616 22 IL-15 hybrid N/A 23 IL-10R TRAP (IL-10 antagonist) [alpha] NP_001549 24 IL-10R Trap N/A 25 IL-27R TRAP (IL-27 antagonist) NP_004834 26 IL-13 AAH96140 27 IL-17 AAC50341 28 IL-33 Isoform a NP_001300974 29 IL-33 Isoform a NP_001300973 30 IL-33 Isoform b NP_001186569 31 IL-33 Isoform c NP_001186570 32 IL-33 Isoform d NP_001300975; NP_001300976 33 IL-33 Isoform e NP_001300977 34 IFN-gamma AAB59534 35 secreted flagellin N/A 36 anti-CTLA4 (CTLA-4 antagonist N/A antibody) 38 Interleukin-2 (IL-2) (murine) NM_008366.3 39 Xc11 (murine) GenBank: BC062249.1 42 4-1BBL (TNFSF9) (murine) GenBank: BC138767.1 55 IL-15Rα N/A 58 hαCTLA4 N/a com1-19 Anti-PD-1 (PD-1 antagonist antibody) Anti-PD-L1 (PD-L1 antagonist antibody) Anti-TIGIT (TIGIT antagonist antibody) Anti-TGFβ (TGFβ antagonist antibody) Anti-IL6R (IL-6 receptor antibody)

Additional detail about the above-discussed agonists that can be expressed from an adenovirus vector as described herein is provided below: GITR Ligand.

In one embodiment, the heterologous gene is a GITR ligand family gene, such as TNFSF18 (also known as GITRL) (See, e.g., Tone, M., Tone, Y., Adams, E., Yates, S. F., Frewin, M. R., Cobbold, S. P., & Waldmann, H. (2003). Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells. Proceedings of the National Academy of Sciences of the United States of America, 100(25), 15059-15064. doi:10.1073/pnas.2334901100). The GITR/GITRL signaling pathway is associated with activation of immune cells Nocentini, G., Ronchetti, S., Petrillo, M. G., & Riccardi, C. (2012). Pharmacological modulation of GITRL/GITR system: therapeutic perspectives. British Journal of Pharmacology, 165(7), 2089-99. doi:10.1111/j.1476-5381.2011.01753.x). Specifically, the GITRL protein has an immunomodulatory activity including inhibiting the suppressive activity of T regulatory cells and activation of T effector cells. The intratumoral localization of effective amounts of GITRL protein results in stimulation of the immune system and inhibition of the tumor, resulting in more effective viral-based therapeutic treatment of human subjects suffering from cancer. Moreover, the costimulatory activity of GITRL protein has a synergistic effect with the tumor-directed cell-lytic activity of the adenovirus, resulting from activation and/or recruitment of the immune system to the tumor and enhanced antigen presentation IL-10 Trap.

In one embodiment, the heterologous gene is an engineered IL-10 trap, such as IL-10 receptor fused to a human immunoglobulin Fc domain. Examples include IL10RA-Fc fusion protein or IL10RA-IL10RB-Fc fusion protein. (See, e.g., Economides, A. N., Carpenter, L. R., Rudge, J. S., Wong, V., Koehler-Stec, E. M., Hartnett, C., . . . Stahl, N. (2003). Cytokine traps: multi-component, high-affinity blockers of cytokine action. Nature Medicine, 9(1), 47-52. doi:10.1038/nm811). The IL-10 family is associated with inhibition of inflammatory response in immune cells through inhibition of the expression of proinflammatory cytokines and co-stimulatory molecules. Expression of IL-10 by T regulatory cells suppresses the activity of T effector cells (Mosser D M, Zhang X. Immunol Rev. 2008 December; 226:205-18. Interleukin-10: new perspectives on an old cytokine). Specifically, the IL-10 trap protein has an immunomodulatory activity by inhibiting the anti-inflammatory activity of IL-10. The intratumoral localization of effective amounts of IL-10 trap protein results in stimulation of the immune system and inhibition of the tumor, resulting in more effective viral-based therapeutic treatment of human subjects suffering from cancer. Moreover, IL-10 trap protein inhibition of IL-10's anti-inflammatory activity has a synergistic effect with the tumor-directed cell-lytic activity of the adenovirus, resulting from activation and/or recruitment of the immune system to the tumor and enhanced antigen presentation. In one embodiment, the IL-10 Receptor Trap includes all or a portion of the extracellular domains of IL-10Rα and IL-10Rα.

Anti-CTLA4.

In one embodiment, the heterologous gene is an antibody (or domain or fragment thereof) that inhibits the function of CTLA4 (See, e.g., Leach D R, Krummel M F, Allison J P Enhancement of antitumor immunity by CTLA-4 blockade. Science. 1996 Mar. 22; 271(5256):1734-6.) The CTLA4 family is associated with inhibition of T cells through the interaction with ligands CD80 and CD86 (Krummel M F, Allison J P (1995). “CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation”. J. Exp. Med. 182 (2): 459-65.) Specifically, the anti-CTLA4 antibody has an immunomodulatory activity including blocking the inhibitory function of CTLA4 resulting in more efficient activation of T effector cells. The intratumoral localization of effective amounts of anti-CTLA4 antibody results in stimulation of the immune system and inhibition of the tumor, resulting in more effective viral-based therapeutic treatment of human subjects suffering from cancer. Moreover, anti-CTLA4 inhibition of CTLA4's T cell inhibitory activity has a synergistic effect with the tumor-directed cell-lytic activity of the adenovirus, resulting from activation and/or recruitment of the immune system to the tumor and enhanced antigen presentation.

IL-12:

In one embodiment, the heterologous gene is a member of the Interleukin cytokine family such as IL-12. The IL-12 cytokine family is associated with induction of IFNγ and mediating T-cell dependent immunity. Specifically, IL-12 is an immunostimulatory cytokine with strong antiangiogenic effects. IL-12 has immunomodulatory activity including cell proliferation, lymphocyte differentiation and NK cell activation. The intratumoral localization of effective amounts of IL-12 results in the differentiation, proliferation, and maintenance of T helper 1 (Th1) responses that lead to IFNγ and IL-2 production that in turn, promote T cell responses and macrophage activation. The local expression of effective amounts of IL-12 from intratumoral injections may provide a safety benefit over systemic administration and side effects associated with high IL-12 serum levels. Moreover, the immunostimulatory activity and induction of cytotoxicity mediated by natural killer cells and T cells by IL-12 may have a synergistic effect with the tumor-directed cell-lytic and immune stimulating activity of our adenovirus providing a more effective viral-based therapeutic treatment of human subjects suffering from cancer. In one embodiment, the IL-12 polypeptide is a fusion of IL-12 subunits p35 and p40, linked by a 45 bp linker, including IL-1213:p40 (NM_001303244) and IL-12 Alpha:p35 (NM_008351.1).

GM-CSF:

In one embodiment, the heterologous gene is a cytokine such as Granulocyte-macrophage colony-stimulating factor (GM-CSF), also known as colony stimulating factor 2 (CSF2). Cytokines are secreted proteins or peptides that mediate and regulate immunity and inflammation. Specifically, GM-CSF has an immunomodulatory activity of functioning as an immune adjuvant and facilitates development of the immune system, acting as a growth factor for DCs and APCs. The localized secretion of effective amounts of GM-CSF results in an increase in dendritic cell (DC) maturation and function as well as macrophage activity, recruiting immune cells to the inflammatory site of tumor treatment, resulting in more effective therapeutic treatment of human subjects suffering from cancer. Moreover, GM-CSF has been demonstrated to be capable of induced long-lasting, specific anti-tumor immunity when combined with cancer vaccines, potentially providing a synergistic effect with the tumor-directed cell-lytic activity of our adenovirus. In a recent phase III clinical trial, an oncolytic herpes simplex virus armed with GM-CSF (T-VEC) showed durable response rates in advanced melanoma patients compared with GM-CSF protein alone.

Secreted Flagellin:

In one embodiment, the heterologous gene comes from a gram-negative bacterium in the Salmonellae family, such as the gene encoding flagellin. Bacterial proteins, including flagellin, are associated with the activation of the innate immune response, leading to production of proinflammatory cytokines and the up-regulation of costimulatory molecules. Specifically, flagellin is a TLR5 agonist, and binding of secreted flagellin to TLR5 stimulates production of TNFα, and induces infiltration of APC's and TIL's to the local tumor environment. By acting as a strong adjuvant, flagellin is able to prime the immune system to elicit strong adaptive immune responses, resulting in enhanced and broadened immune response a more effective viral-based therapeutic treatment of human subjects suffering from cancer. In some embodiments, secreted Flagellin contains a murine IL-2 signal sequence (See NM_008366) and Salmonella Flagellin, GenBank: D13689.

TNFα and non-cleavable TNFα.

In one embodiment, the heterologous gene is an engineered TNFα ligand family gene, such as a non-cleavable (membrane-bound, transmembrane) form of TNFα (See, e.g., Li Q, Li L, Shi W, Jiang X, Xu Y, Gong F, Zhou M, Edwards C K 3rd, Li Z., Mechanism of action differences in the antitumor effects of transmembrane and secretory tumor necrosis factor-alpha in vitro and in vivo. Cancer Immunol Immunother. 2006: 55, 1470-9.). TNFα belongs to a family of pro-inflammatory cytokines (Calcinotto A, Grioni M, Jachetti E, Curnis F, Mondino A, Parmiani G, Corti A, Bellone M. Targeting TNFα to neoangiogenic vessels enhances lymphocyte infiltration in tumors and increases the therapeutic potential of immunotherapy. J Immunol. 2012; 188: 2687-94.). Specifically, expression of TNFα in the tumor microenvironment is expected to increase the inflammatory milieu resulting in increased anti-tumor immune responses. Use of a non-cleavable TNFα results in a tethered form of TNFα which remains membrane-bound. The intratumoral localization of effective amounts of TNFα protein results in stimulation of the immune system and inhibition of growth of the tumor, resulting in more effective viral-based therapeutic treatment of human subjects suffering from cancer. In addition, local expression may provide a safety benefit over systemic administration of TNFα. Moreover, the immunomodulatory activity of the membrane-bound TNFα protein has a synergistic effect with the tumor-directed cell-lytic activity of the adenovirus, resulting from activation and/or recruitment of the immune system to the tumor and enhanced antigen presentation.

OX40L.

In one embodiment, the heterologous gene is an OX40L family gene, such as TNFSF4 (also called OX40L, CD252) (See, e.g., Dannull J, Nair S, Su Z, Boczkowski D, DeBeck C, Yang B, Gilboa E, Vieweg J Enhancing the immunostimulatory function of dendritic cells by transfection with mRNA encoding OX40 ligand. Blood. 2005, 105: 3206-13.). The TNFSF is associated with activation of immune cells (Croft M, So T, Duan W, Soroosh P. The significance of OX40 and OX40L to T-cell biology and immune disease. Immunol Rev. 2009; 229:173-91.). Specifically, OX40L is a co-stimulatory ligand for TNFRSF4 (OX40, CD134) resulting in activation of T cells. Expression of OX40L in the tumor microenvironment and binding to its cognate receptor (OX40) is expected to increase the activity (proliferation, cytokine release) of tumor infiltrating lymphocytes (TILs) resulting in antitumor activity. The intratumoral localization of effective amounts of OX40L protein results in stimulation of the immune system and inhibition of growth of the tumor, resulting in more effective viral-based therapeutic treatment of human subjects suffering from cancer. Local expression may provide a safety benefit over systemic administration of OX40L. Moreover, the immunomodulatory activity of the OX40L protein has a synergistic effect with the tumor-directed cell-lytic activity of the adenovirus, resulting from activation and/or recruitment of the immune system to the tumor and enhanced antigen presentation IL-7.

In one embodiment, the heterologous gene is an IL-7 cytokine family gene, such as IL-7 (See, e.g., Gao J, Zhao L, Wan Y Y, Zhu B., Mechanism of Action of IL-7 and Its Potential Applications and Limitations in Cancer Immunotherapy. Int J Mol Sci. 2015, 16: 10267-80.). IL-7 belongs to a family of pro-inflammatory cytokines (Geiselhart L A, Humphries C A, Gregorio T A, Mou S, Subleski J, Komschlies K L. IL-7 administration alters the CD4:CD8 ratio, increases T cell numbers, and increases T cell function in the absence of activation. J Immunol. 2001; 166: 3019-27.). Specifically, expression of IL-7 in the tumor microenvironment is expected to increase the inflammatory milieu resulting in increased anti-tumor immune responses. The intratumoral localization of effective amounts of IL-7 protein results in stimulation of the immune system and inhibition of growth of the tumor, resulting in more effective viral-based therapeutic treatment of human subjects suffering from cancer. Moreover, the immunomodulatory activity of the IL-7 protein has a synergistic effect with the tumor-directed cell-lytic activity of the adenovirus, resulting from activation and/or recruitment of the immune system to the tumor and enhanced antigen presentation.

CD40L

In one embodiment, the heterologous gene is a member of the TNF superfamily, such as CD40L (also known as CD40LG or CD154) (See, e.g., Hassan G S, et al., 2015. “Role of CD154 in cancer pathogenesis and immunotherapy.” Cancer Treat Rev 4 1(5):431-40). The CD40L is the ligand for CD40 expressed on antigen presenting cells. Specifically, the CD40L protein has a costimulatory activity important for activation of T cell dependent immune responses (Sotomayor E M, et al., 1999. “Conversion of tumor-directed CD4+ T-cell tolerance to T-cell priming through in vivo ligation of CD40.” Nat Med. 5:780-787; French R R, et al., 1999. “CD40 antibody evokes a cytotoxic T-cell response that eradicates lymphoma and bypasses T-cell help. Nat Med. 5:548-553). The intratumoral localization of effective amounts of CD40 protein results in activation of the immune system and lysis of the tumor, resulting in more effective viral-based therapeutic treatment of human subjects suffering from cancer. Moreover, the costimulatory immunomodulatory activity of CD40 protein has a synergistic effect with the tumor-directed cell-lytic activity of the adenovirus, resulting in a local and systemic immune response against the tumor.

IL-15 and IL-15 Hybrid

In one embodiment, the heterologous gene is a cytokine family gene, such as Interleukin 15 (IL-15) (See, e.g., Di Sabatino A, et. al., 2011 “Role of IL-15 in immune-mediated and infectious diseases”. Cytokine Growth Factor Rev. 22 (1): 19-33.; Steel J C, et al., 2012, “Interleukin-15 biology and its therapeutic implications in cancer”. Trends Pharmacol. Sci. 33 (1): 35-41). IL-15 is a cytokine that regulates T cell and NK cell activation and proliferation. (Waldmann T A, et al., (1999). “The multifaceted regulation of interleukin-15 expression and the role of this cytokine in NK cell differentiation and host response to intracellular pathogens”. Annu. Rev. Immunol. 17: 19-49). Specifically, the IL-15 protein has an immunomodulatory activity by providing survival signals to maintain memory T cells in the absence of antigen. IL-15 has also been shown to enhance the anti-tumor immunity of CD8+ T cells (See, Klebanoff C A, et al., “IL-15 enhances the in vivo antitumor activity of tumor-reactive CD8+ T Cells” Proc. Natl. Acad. Sci. USA. 101 (7): 1969-74; and Teague R M, et al., “Interleukin-15 rescues tolerant CD8+ T cells for use in adoptive immunotherapy of established tumors” Nat. Med. 12 (3): 335-41). Expression of an IL-15 hybrid molecule (IL-15 linked to the IL-15 Receptor alpha, see Tosic et al., PLos ONE 9(10): e109801 (2014)) leads to stabilization and increased bioactivity (Bergamaschi et al., 2008. “Intracellular Interaction of Interleukin-15 with Its Receptor a during Production Leads to Mutual Stabilization and Increased Bioactivity”, JBC, 283(7):4189-99; Bergamaschi et al., 2013. “Circulating IL-15 exists as heterodimeric complex with soluble IL-15Ra in human and mouse serum”, Blood 120(1):e1). The intratumoral localization of effective amounts of IL-15 protein results in activation of the immune system and lysis of the tumor, resulting in more effective viral-based therapeutic treatment of human subjects suffering from cancer. Moreover, the survival signals provided by IL-15 protein has a synergistic effect with the tumor-directed cell-lytic activity of the adenovirus, resulting in a local and systemic immune response against the tumor. In some embodiments, an IL-15 hybrid includes IL-15, P2A, and IL-15Ra, IL-15 (NM_008357), 68 bp P2A, and IL-15Rα (GenBank: BC132233.1).

CD70

In one embodiment, the heterologous gene is a member of the TNF superfamily, such as CD70 (also known as TNFSF7 or CD27L). (See, e.g., Denoeud J and Moser M., 2011. “Role of CD27/CD70 pathway of activation in immunity and tolerance.” J Leukoc Biol. 89(2):195-203). CD70 is expressed on activated T and B cells, as well as mature dendritic cells, and acts as a ligand for CD27. CD70 plays a costimulatory role in promoting T cell expansion and differentiation (Keller A M, et al., 2008. “Expression of costimulatory ligand CD70 on steady-state dendritic cells breaks CD8+ T cell tolerance and permits effective immunity,” Immunity 29(6):934-46; Bonehill A, et al., 2008 “Enhancing the T-cell stimulatory capacity of human dendritic cells by co-electroporation with CD40L, CD70 and constitutively active TLR4 encoding mRNA,” Mol Ther. 16(6):1170-80). In addition, CD70 expression in the tumor microenvironment will increase NK-mediated tumor clearance and promote an adaptive immune response against the tumor (Kelly J M, et al., 2002. “Induction of tumor-directed T cell memory by NK cell-mediated tumor rejection,” Nat Immunol. 3(1):83-90). The intratumoral localization of effective amounts of CD70 protein results in activation of the immune system and lysis of the tumor, resulting in more effective viral-based therapeutic treatment of human subjects suffering from cancer. Moreover, the immunomodulatory activity of CD70 protein has a synergistic effect with the tumor-directed cell-lytic activity of the adenovirus, resulting in a local and systemic immune response against the tumor.

In a specific embodiment, the agonist of a co-stimulatory signal of an immune cell expressed by the adenovirus is an agonist of a co-stimulatory receptor expressed by an immune cell. Specific examples of co-stimulatory receptors that can be expressed by the adenovirus include glucocorticoid-induced tumor necrosis factor receptor (GITR), Inducible T-cell co-stimulator (ICOS or CD278), OX40 (CD134), CD27, CD28, 4-IBB(CD137), CD40, CD226, cytotoxic and regulatory T cell molecule (CRT AM), death receptor 3 (DR3), lymphotoxin-beta receptor (LTBR), transmembrane activator and CAML interactor (TACI), B cell-activating factor receptor (BAFFR), and B cell maturation protein (BCMA). In a specific embodiment, the agonist of a co-stimulatory receptor expressed by an immune cell is an antibody (or an antigen-binding fragment thereof) or ligand that specifically binds to the co-stimulatory receptor. In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is an sc-Fv. In a specific embodiment, the antibody is a bispecific antibody that binds to two receptors on an immune cell. In one embodiment, the bispecific antibody binds to a receptor on an immune cell and to another receptor on a cancer cell. In specific embodiments, the antibody is a human or humanized antibody. In certain embodiments, the ligand or antibody is a chimeric protein.

In a specific embodiment, the antagonist of an inhibitory signal of an immune cell is an antagonist of an inhibitory receptor expressed by an immune cell. Specific examples of inhibitory receptors include cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4 or CD52), programmed cell death protein 1 (PD1 or CD279), B and T-lymphocyte attenuator (BTLA), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene 3 (LAG3), T-cell membrane protein 3 (TIM3), adenosine A2a receptor (A2aR), T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), and CD160. In a specific embodiment, the antagonist of an inhibitory receptor expressed by an immune cell is an antibody (or an antigen-binding fragment thereof) that specifically binds to the co-stimulatory receptor.

Antibodies include, but are not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, single domain antibodies, camelid or camelized antibodies, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab′) fragments, disulfide-linked bispecific Fvs (sdFv), intrabodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Id antibodies to antibodies), and epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. In a specific embodiment, an antibody is a human or humanized antibody. In another specific embodiment, an antibody is a monoclonal antibody or scFv. In certain embodiments, an antibody is a human or humanized monoclonal antibody or scFv. In other specific embodiments, the antibody is a bispecific antibody. In certain embodiments, the bispecific antibody specifically binds to a co-stimulatory receptor of an immune cell or an inhibitory receptor of an immune, and a receptor on a cancer cell. In some embodiments, the bispecific antibody specifically binds to two receptors immune cells, e.g., two co-stimulatory receptors on immune cells, two inhibitory receptors on immune cells, or one co-stimulatory receptor on immune cells and one inhibitory receptor on immune cells.

The recombinant AVs described herein may be engineered to express any agonist of a co-stimulatory signal and/or any antagonist of an inhibitory signal of an immune cell, such as, e.g., a T-lymphocyte, NK cell or antigen-presenting cell (e.g., a dendritic cell or macrophage). In specific embodiments, the agonist and/or antagonist is an agonist of a human co-stimulatory signal of an immune cell and/or antagonist of a human inhibitory signal of an immune cell. In certain embodiments, the agonist of a co-stimulatory signal is an agonist of a co-stimulatory molecule (e.g., co-stimulatory receptor) found on immune cells, such as, e.g., T-lymphocytes (e.g., CD4+ or CD8+ T-lymphocytes), NK cells and/or antigen-presenting cells (e.g., dendritic cells or macrophages). Specific examples of co-stimulatory molecules include glucocorticoid-induced tumor necrosis factor receptor (GITR), Inducible T-cell co-stimulator (ICOS or CD278), OX40 (CD134), CD27, CD28, 4-IBB (CD137), CD40, lymphotoxin alpha (LT alpha), LIGHT (lymphotoxin-like, exhibits inducible expression, and competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes), CD226, cytotoxic and regulatory T cell molecule (CRT AM), death receptor 3 (DR3), lymphotoxin-beta receptor (LTBR), transmembrane activator and CAML interactor (TACI), B cell-activating factor receptor (BAFFR), and B cell maturation protein (BCMA). In specific embodiments, the agonist is an agonist of a human co-stimulatory receptor of an immune cell. In certain embodiments, the agonist of a co-stimulatory receptor is not an agonist of ICOS. In some embodiments, the antagonist is an antagonist of an inhibitory molecule (e.g., inhibitory receptor) found on immune cells, such as, e.g., T-lymphocytes (e.g., CD4+ or CD8+ T-lymphocytes), NK cells and/or antigen-presenting cells (e.g., dendritic cells or macrophages). Specific examples of inhibitory molecules include cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4 or CD52), programmed cell death protein 1 (PD1 or CD279), B and T-lymphocyte attenuator (BTLA), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene 3 (LAG3), T-cell membrane protein 3 (TIM3), CD 160, adenosine A2a receptor (A2aR), T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), and CD 160. In specific embodiments, the antagonist is an antagonist of a human inhibitory receptor of an immune cell.

In a specific embodiment, the agonist of a co-stimulatory receptor is an antibody or antigen-binding fragment thereof that specifically binds to the co-stimulatory receptor. Specific examples of co-stimulatory receptors include GITR, ICOS, OX40, CD27, CD28, 4-1BB, CD40, LT alpha, LIGHT, CD226, CRT AM, DR3, LTBR, TACI, BAFFR, and BCMA. In certain specific embodiments, the antibody is a monoclonal antibody. In other specific embodiments, the antibody is an sc-Fv. In a specific embodiment, the antibody is a bispecific antibody that binds to two receptors on an immune cell. In other embodiments, the bispecific antibody binds to a receptor on an immune cell and to another receptor on a cancer cell. In specific embodiments, the antibody is a human or humanized antibody.

In another embodiment, the agonist of a co-stimulatory receptor expressed by the adenovirus is a ligand of the co-stimulatory receptor. In certain embodiments, the ligand is fragment of a native ligand. Specific examples of native ligands include ICOSL, B7RP1, CD137L, OX40L, CD70, herpes virus entry mediator (HVEM), CD80, and CD86. The nucleotide sequences encoding native ligands as well as the amino acid sequences of native ligands are known in the art. For example, the nucleotide and amino acid sequences of B7RP1 (otherwise known as ICOSL; GenBank human: NM_015259.4, NP_056074.1 murine: NM_015790.3, NP_056605.1), CD137L(GenBank human: NM_003811.3, NP_003802.1, murine: NM_009404.3, NP_033430.1), OX40L(GenBank human: NM_003326.3, NP_003317.1, murine: NM_009452.2, NP_033478.1), CD70(GenBank human: NM_001252.3, NP_001243.1, murine: NM_011617.2, AAD00274.1), CD80(GenBank human: NM_005191.3, NP_005182.1, murine: NM_009855.2, NP_033985.3), and CD86 (GenBank human: NM_005191.3, CAG46642.1, murine: NM_019388.3, NP_062261.3) can be found in GenBank. In other embodiments, the ligand is a derivative (e.g., a fragment, domain, fusion, or other modification of a full-length polypeptide) a native ligand. In some embodiments, the ligand is a fusion protein comprising at least a portion of the native ligand or a derivative of the native ligand that specifically binds to the co-stimulatory receptor, and a heterologous amino acid sequence. In specific embodiments, the fusion protein comprises at least a portion of the native ligand or a derivative of the native ligand that specifically binds to the co-stimulatory receptor, and the Fc portion of an immunoglobulin or a fragment thereof. An example of a ligand fusion protein is a 4-IBB ligand fused to Fc portion of immunoglobulin (described by Meseck M et al., J Immunother. 2011 34: 175-82).

In another embodiment, the antagonist of an inhibitory receptor expressed by the adenovirus is an antibody (or an antigen-binding fragment) or a soluble receptor that specifically binds to the native ligand for the inhibitory receptor and blocks the native ligand from binding to the inhibitory receptor and transducing an inhibitory signal(s). Specific examples of native ligands for inhibitory receptors include PDL-1, PDL-2, B7-H3, B7-H4, HVEM, Gal9 and adenosine. Specific examples of inhibitory receptors that bind to a native ligand include CTLA-4, PD-1, BTLA, KIR, LAG3, TIM3, and A2aR.

In specific embodiments, the antagonist of an inhibitory receptor expressed by the adenovirus is a soluble receptor that specifically binds to the native ligand for the inhibitory receptor and blocks the native ligand from binding to the inhibitory receptor and transducing an inhibitory signal(s). In certain embodiments, the soluble receptor is a fragment of a native inhibitory receptor or a fragment of a derivative of a native inhibitory receptor that specifically binds to native ligand {e.g., the extracellular domain of a native inhibitory receptor or a derivative of an inhibitory receptor). In some embodiments, the soluble receptor is a fusion protein comprising at least a portion of the native inhibitory receptor or a derivative of the native inhibitory receptor (e.g., the extracellular domain of the native inhibitory receptor or a derivative of the native inhibitory receptor), and a heterologous amino acid sequence. In specific embodiments, the fusion protein comprises at least a portion of the native inhibitory receptor or a derivative of the native inhibitory receptor, and the Fc portion of an immunoglobulin or a fragment thereof. An example of a soluble receptor fusion protein is a LAG3-Ig fusion protein (described by Huard B et al, Eur J Immunol (1995) 25:2718-21).

In specific embodiments, the antagonist of an inhibitory receptor expressed by the adenovirus is an antibody (or an antigen-binding fragment) that specifically binds to the native ligand for the inhibitory receptor and blocks the native ligand from binding to the inhibitory receptor and transducing an inhibitory signal(s). In certain specific embodiments, the antibody is a monoclonal antibody. In other specific embodiments, the antibody is an scFv. In particular embodiments, the antibody is a human or humanized antibody. A specific example of an antibody to inhibitory ligand is anti-PD-L1 antibody (Iwai Y, et al. PNAS 2002; 99: 12293-12297).

In another embodiment, the antagonist of an inhibitory receptor expressed by the adenovirus is an antibody (or an antigen-binding fragment) or ligand that binds to the inhibitory receptor, but does not transduce an inhibitory signal(s). Specific examples of inhibitory receptors include CTLA-4, PD1, BTLA, TIGIT, KIR, LAG3, TIM3, and A2aR. In certain specific embodiments, the antibody is a monoclonal antibody. In other specific embodiments, the antibody is an scFv. In particular embodiments, the antibody is a human or humanized antibody. A specific example of an antibody to inhibitory receptor is anti-CTLA-4 antibody (Leach D R, et al. Science 1996; 271: 1734-1736). Another example of an antibody to inhibitory receptor is anti-PD-1 antibody (Topalian S L, NEJM 2012; 28:3167-75).

In certain embodiments, a chimeric adenovirus described herein is engineered to produce an antagonist of CTLA-4, such as, e.g., ipilimumab or tremelimumab. In certain embodiments, a chimeric adenovirus described herein is engineered to an antagonist of PD1, such as, e.g., MDX-1106 (BMS-936558), MK3475, CT-011, AMP-224, or MDX-1105. In certain embodiments, a chimeric adenovirus described herein is engineered to express an antagonist of LAG3, such as, e.g., IMP321. In certain embodiments, a chimeric adenovirus described herein is engineered to express an antibody (e.g., a monoclonal antibody or an antigen-binding fragment thereof, or scFv) that binds to B7-H3, such as, e.g., MGA271. In specific embodiments, a chimeric adenovirus described herein is engineered to express an agonist of a co-stimulatory signal of an immune cell and/or an antagonist of an inhibitory signal of an immune cell. In specific embodiments, adenovirus described herein is engineered to express anti-CD28 scFv, ICOSL, CD40L, OX40L, CD137L, GITRL, and/or CD70.

In certain embodiments, an agonist of a co-stimulatory signal of an immune cell expressed by the adenovirus induces (e.g., selectively) induces one or more of the signal transduction pathways induced by the binding of a co-stimulatory receptor to its ligand. In specific embodiments, an agonist of a co-stimulatory receptor induces one or more of the signal transduction pathways induced by the binding of the co-stimulatory receptor to one or more of its ligands by at least 25%, 30%, 40%>, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in the range of between 25% to 50%, 25% to 75%, 50% to 75%, 50% to 95%, 75% to 95%, or 75% to 100% relative to the one or more signal transduction pathways induced by the binding of the co-stimulatory receptor to one or more of its ligands in the absence of the agonist. In specific embodiments, an agonist of a co-stimulatory receptor: (i) induces one or more of the signal transduction pathways induced by the binding of the co-stimulatory receptor to one particular ligand by at least 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in the range of between 25% to 50%, 25% to 75%, 50% to 75%, 50% to 95%, 75% to 95%, or 75% to 100% relative to the one or more signal transduction pathways induced by the binding of the co-stimulatory receptor to the particular ligand in the absence of the agonist; and (ii) does not induce, or induces one or more of the signal transduction pathways induced by the binding of the co-stimulatory receptor to one or more other ligands by less than 20%, 15%, 10%, 5%, or 2%, or in the range of between 2% to 5%, 2% to 10%, 5% to 10%, 5% to 15%, 5% to 20%, 10% to 15%, or 15% to 20% relative to the one or more signal transduction pathways induced by the binding of the co-stimulatory receptor to such one or more other ligands in the absence of the agonist.

In certain embodiments, an agonist of a co-stimulatory signal of an immune cell activates or enhances (e.g., selectively activates or enhances) one or more of the signal transduction pathways induced by the binding of a co-stimulatory receptor to its ligand. In specific embodiments, an agonist of a co-stimulatory receptor activates or enhances one or more of the signal transduction pathways induced by the binding of the co-stimulatory receptor to one or more of its ligands by at least 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in the range of between 25% to 50%, 25% to 75%, 50% to 75%, 50% to 95%, 75% to 95%), or 75%) to 100% relative to the one or more signal transduction pathways induced by the binding of co-stimulatory receptor to one or more of its ligands in the absence of the agonist. In specific embodiments, an agonist of a co-stimulatory receptor: (i) an agonist of a co-stimulatory signal activates or enhances one or more of the signal transduction pathways induced by the binding of the co-stimulatory receptor to one particular ligand by at least 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in the range of between 25% to 50%, 25% to 75%, 50% to 75%, 50% to 95%, 75% to 95%, or 75% to 100% relative to the one or more signal transduction pathways induced by the binding of the co-stimulatory receptor to the particular ligand in the absence of the agonist; and (ii) does not activate or enhance, or activates or enhances one or more of the signal transduction pathways induced by the binding of the co-stimulatory receptor to one or more other ligands by less than 20%, 15%, 10%, 5%, or 2%, or in the range of between 2% to 5%, 2% to 10%, 5% to 10%, 5% to 15%, 5% to 20%, 10% to 15%, or 15% to 20% relative to the one or more signal transduction pathways induced by the binding of the co-stimulatory receptor to such one or more other ligands in the absence of the agonist.

In some embodiments, an antagonist of an inhibitory signal of an immune cell (e.g., selectively) inhibits or reduces one or more of the signal transduction pathways induced by the binding of an inhibitory receptor to its ligand. In specific embodiments, an antagonist of an inhibitory receptor inhibits or reduces one or more of the signal transduction pathways induced by the binding of the inhibitory receptor to one or more of its ligands by at least 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in the range of between 25% to 50%, 25% to 75%, 50% to 75%, 50% to 95%, 75% to 95%, or 75% to 100% relative to the one or more signal transduction pathways induced by the binding of the inhibitory receptor to one or more of its ligands in the absence of the antagonist. In specific embodiments, an antagonist of an inhibitory receptor: (i) inhibits or reduces one or more of the signal transduction pathways induced by the binding of the inhibitory receptor to one particular ligand by at least 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in the range of between 25% to 50%, 25% to 75%, 50% to 75%, 50% to 95%, 75% to 95%, or 75% to 100% relative to the one or more signal transduction pathways induced by the binding of the inhibitory receptor to the one particular ligand in the absence of the antagonist; and (ii) does not inhibit or reduce, or inhibits or reduces one or more of the signal transduction pathways induced by the binding of the inhibitory receptor to one or more other ligands by less than 20%, 15%, 10%, 5%, or 2%, or in the range of between 2% to 5%, 2% to 10%, 5% to 10%, 5% to 15%, 5% to 20%, 10% to 15%, or 15% to 20% relative to the one or more signal transduction pathways induced by the binding of inhibitory receptor to such one or more other ligands in the absence of the antagonist.

In specific embodiments, an agonist of a co-stimulatory signal of an immune cell and/or an antagonist of an inhibitory signal of an immune cell induces, activates and/or enhances one or more immune activities, functions or responses. The one or more immune activities, functions or responses can be in the form of, e.g., an antibody response (humoral response) or a cellular immune response, e.g., cytokine secretion (e.g., interferon-gamma), helper activity or cellular cytotoxicity. In one embodiment, expression of an activation marker on immune cells (e.g., CD44, Granzyme, or Ki-67), expression of a co-stimulatory receptor on immune cells (e.g., ICOS, CD28, OX40, or CD27), expression of a ligand for a co-stimulatory receptor (e.g., B7HRP1, CD80, CD86, OX40L, or CD70), cytokine secretion, infiltration of immune cells (e.g., T-lymphocytes, B lymphocytes and/or NK cells) to a tumor, antibody production, effector function, T cell activation, T cell differentiation, T cell proliferation, B cell differentiation, B cell proliferation, and/or NK cell proliferation is induced, activated and/or enhanced following contact with an agonist of a co-stimulatory signal of an immune cell and/or an antagonist of an inhibitory signal of an immune cell. In another embodiment, myeloid-derived suppressor cell (MDSC) tumor infiltration and proliferation, Treg tumor infiltration, activation and proliferation, peripheral blood MDSC and Treg counts are inhibited following contact with an agonist of a co-stimulatory signal of an immune cell and/or an antagonist of an inhibitory signal of an immune cell.

In certain embodiments, a chimeric adenovirus described herein is engineered to produce two or more immunomodulatory polypeptides. In some embodiments, the chimeric adenovirus produces a first immunomodulatory polypeptide and a second immunomodulatory polypeptide.

For example, a first immunomodulatory polypeptide is a costimulatory ligand, a proinflammatory cytokine, an inhibitor of an inhibitory cytokine, an initiator of a localized immune response, an inhibitor of a co-inhibitory checkpoint molecule, or a ligand of a cluster of differentiation (CD) molecule, and the second immunomodulatory polypeptide is a costimulatory ligand, a proinflammatory cytokine, an inhibitor of an inhibitory cytokine, an initiator of a localized immune response, an inhibitor of a co-inhibitory checkpoint molecule, or a ligand of a cluster of differentiation (CD) molecule.

For example, a first immunomodulatory polypeptide is a costimulatory ligand, and the second immunomodulatory polypeptide is a costimulatory ligand.

For another example, a first immunomodulatory polypeptide is a costimulatory ligand and the second immunomodulatory polypeptide is a pro-inflammatory cytokine.

In some embodiments, two or more immunomodulatory polypeptides are expressed from a single transcript. To express two or more proteins from a single transcript determined by a viral or non-viral vector, an internal ribosome entry site (IRES) sequence is commonly used to drive expression of the second, third, fourth coding sequence, etc. When two coding sequences are linked via an IRES, the translational expression level of the second coding sequence is often significantly reduced (Furler et al. 2001. Gene Therapy 8:864-873). In fact, the use of an IRES to control transcription of two or more coding sequences operably linked to the same promoter can result in lower level expression of the second, third, etc. coding sequence relative to the coding sequence adjacent the promoter. In addition, an IRES sequence may be sufficiently long to impact complete packaging of the vector, e.g., the eCMV IRES has a length of 507 base pairs.

Internal ribosome entry site (IRES) elements were first discovered in picornavirus mRNAs (Jackson et al. 1990. Trends Biochem. Sci. 15:477-83) and Jackson and Kaminski, RNA (1995) 1:985-1000). Examples of IRES generally employed by those of skill in the art include those referenced in Table I and Appendix A, as well as those described in U.S. Pat. No. 6,692,736. Examples of “IRES” known in the art include, but are not limited to IRES obtainable from picornavirus (Jackson et al., 1990) and IRES obtainable from viral or cellular mRNA sources, such as for example, immunoglobulin heavy-chain binding protein (BiP), the vascular endothelial growth factor (VEGF) (Huez et al. 1998, Mol. Cell. Biol. 18:6178-6190), the fibroblast growth factor 2 (FGF-2), and insulin-like growth factor (IGFII), the translational initiation factor eIF4G and yeast transcription factors TFIID and HAP4, the encephelomyocarditis virus (EMCV) which is commercially available from Novagen (Duke et al. 1992. J. Virol 66:1602-9) and the VEGF IRES (Huez et al. 1998. Mol. Cell. Biol. 18:6178-90). IRES have also been reported in different viruses such as cardiovirus, rhinovirus, aphthovirus, HCV, Friend murine leukemia virus (FrMLV) and Moloney murine leukemia virus (MoMLV). As used herein, “IRES” encompasses functional variations of IRES sequences as long as the variation is able to promote direct internal ribosome entry to the initiation codon of a cistron. An IRES may be mammalian, viral or protozoan. The IRES promotes direct internal ribosome entry to the initiation codon of a downstream cistron, leading to cap-independent translation. Thus, the product of a downstream cistron can be expressed from a bicistronic (or multicistronic) mRNA, without requiring either cleavage of a polyprotein or generation of a monocistronic mRNA. Internal ribosome entry sites are approximately 450 nucleotides in length and are characterized by moderate conservation of primary sequence and strong conservation of secondary structure. The most significant primary sequence feature of the IRES is a pyrimidine-rich site whose start is located approximately 25 nucleotides upstream of the 3′ end of the IRES. See Jackson et al. (1990). Three major classes of picornavirus IRES have been identified and characterized: the cardio- and aphthovirus class (for example, the encephalomyocarditis virus, Jang et al. 1990. Gene Dev 4:1560-1572); the entero- and rhinovirus class (for example, polioviruses, Borman et al. 1994. EMBO J. 13:3149-3157); and the hepatitis A virus (HAV) class, Glass et al. 1993. Virol 193:842-852). For the first two classes, two general principles apply. First, most of the 450-nucleotide sequence of the IRES functions to maintain particular secondary and tertiary structures conducive to ribosome binding and translational initiation. Second, the ribosome entry site is an AUG triplet located at the 3′ end of the IRES, approximately 25 nucleotides downstream of a conserved oligopyrimidine tract. Translation initiation can occur either at the ribosome entry site (cardioviruses) or at the next downstream AUG (entero/rhinovirus class). Initiation occurs at both sites in aphthoviruses. HCV and pestiviruses such as bovine viral diarrhea virus (BVDV) or classical swine fever virus (CSFV) have 341 nt and 370 nt long 5′-UTR respectively. These 5′-UTR fragments form similar RNA secondary structures and can have moderately efficient IRES function (Tsukiyama-Kohara et al. 1992. J. Virol. 66:1476-1483; Frolov et al. 1998. RNA 4:1418-1435). Recent studies showed that both Friend-murine leukemia virus (MLV) 5′-UTR and rat retrotransposon virus-like 30S (VL30) sequences contain IRES structure of retroviral origin (Torrent et al. 1996. Hum. Gene Ther 7:603-612). In eukaryotic cells, translation is normally initiated by the ribosome scanning from the capped mRNA 5′ end, under the control of initiation factors. However, several cellular mRNAs have been found to have IRES structure to mediate the cap-independent translation (van der Velde, et al. 1999. Int J Biochem Cell Biol. 31:87-106). Examples of IRES elements include, without limitation, immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. 1991. Nature 353:90-94), antennapedia mRNA of Drosophila (Oh et al. 1992. Gene and Dev 6:1643-1653), fibroblast growth factor-2 (FGF-2) (Vagner et al. 1995. Mol. Cell. Biol. 15:35-44), platelet-derived growth factor B (PDGF-B) (Bernstein et al. 1997. J. Biol. Chem. 272:9356-9362), insulin-like growth factor II (Teerink et al. (1995) Biochim. Biophys. Acta 1264:403-408), and the translation initiation factor eIF4G (Gan et al. 1996. J. Biol. Chem. 271:623-626). Recently, vascular endothelial growth factor (VEGF) was also found to have IRES element (Stein et al. 1998. Mol. Cell. Biol. 18:3112-3119; Huez et al. 1998. Mol. Cell. Biol. 18:6178-6190). Further examples of IRES sequences include Picornavirus HAV (Glass et al. 1993. Virology 193:842-852); EMCV (Jang and Wimmer. 1990. Gene Dev. 4:1560-1572); Poliovirus (Borman et al. 1994. EMBO J. 13:3149-3157); HCV (Tsukiyama-Kohara et al. 1992. J. Virol. 66:1476-1483); pestivirus BVDV (Frolov et al. 1998. RNA. 4:1418-1435); Leishmania LRV-1 (Maga et al. 1995. Mol. Cell. Biol. 15:4884-4889); Retroviruses: MoMLV (Torrent et al. 1996. Hum. Gene Ther. 7:603-612). VL30, Harvey murine sarcoma virus, REV (Lopez-Lastra et al. 1997. Hum. Gene Ther. 8:1855-1865). IRES may be prepared using standard recombinant and synthetic methods known in the art. For cloning convenience, restriction sites may be engineered into the ends of the IRES fragments to be used.

In some embodiments, two immunomodulatory polypeptides are expressed from separate transcripts, i.e., a first transcript and a second transcript. In some embodiments, the two transcripts are encoded by a DNA insertion at the same location in the adenovirus, e.g., both inserted in E1b, E3, or E4. In some embodiments, the two transcripts are encoded by a DNA insertion at the different locations in the adenovirus, e.g., a first transcript DNA inserted in E1b and a second transcript DNA inserted in E3 or E4, or alternatively, a first transcript DNA inserted in E3 and a second transcript DNA inserted in E4.

Another aspect of the invention provides a virus for causing expression in a target cell of a plurality of recombinant immunomodulatory polypeptides or other protein(s) or polypeptides of interest, wherein the vector also includes a promoter operably linked to a first coding sequence for a first recombinant immunomodulatory polypeptide, a self-processing or other cleavage coding sequence, such as a 2A or 2A-like sequence or a protease recognition site, and a second coding sequence for a second recombinant immunomodulatory polypeptide, wherein the self-processing cleavage sequence or protease recognition site coding sequence is inserted between the first and the second coding sequences. In a related embodiment, the viral vector comprises an expression vector as described above wherein the expression vector further comprises an additional proteolytic cleavage site between the first and second recombinant immunomodulatory polypeptides. A preferred additional proteolytic cleavage site is a furin cleavage site with the consensus sequence RXR/K-R.

Interaction of Oncolytic Activity and Immunomodulatory Activity.

Oncolytic viruses (OVs) were originally conceived as simply a means of targeted destruction of cancer cells. However, it is now thought that the most effective OV therapies will be those that combine tumor cell death with the stimulation of a host anti-tumor immune response. OVs engineered to express particular immunomodulatory cytokines in tumor cells will be able to specifically guide the immune system toward combating cancer cells. Combining the expression of these cytokines with the release of tumor-associated antigens (TAAs, i.e. tumor cell debris) upon viral lysis of cancer cells will allow for the development of cellular or antibody-mediated anti-tumor immune responses (Lichty et al., 2014, Nature Reviews Cancer, 14: 559-567).

Pharmaceutical Compositions.

A pharmaceutical composition of the invention comprises at least one of the vectors of the invention as described herein. Furthermore, the composition may comprise at least two, three or four different (i.e., expressing different transgenes) vectors of the invention. In addition to the vector, a pharmaceutical composition may also comprise any other vectors, such as other adenoviral vectors, other therapeutically effective agents, any other agents such as pharmaceutically acceptable carriers, buffers, excipients, adjuvants, antiseptics, filling, stabilizing or thickening agents, and/or any components, e.g., such as components found in corresponding viral or pharmaceutical products.

The vector(s) described herein can be administered (e.g., in a pharmaceutical composition) to any human or animal, including but not limited to a human or non-human animal having or diagnosed with cancer. According to one embodiment, the cancer is nasopharyngeal cancer, synovial cancer, hepatocellular cancer, renal cancer, cancer of connective tissues, melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia/lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, brain cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, colorectal cancer, rectal cancer, esophagus cancer, gall bladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, oral cancer, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, pancreatic cancer, parathyroid cancer, penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vagina cancer, vulva cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer, tonsil cancer. The vector or pharmaceutical composition of the invention may be administered to any eukaryotic subject selected from a group consisting of animals and human beings, in a preferred embodiment of the invention, the subject is a human or a non-human animal. An animal may be selected from a group consisting of pets, domestic animals and production animals.

The adenoviral vector(s) of the present invention may be administered to a subject, preferably suspended in a biologically compatible solution or pharmaceutically acceptable delivery vehicle. A suitable vehicle includes sterile saline. Other aqueous and non-aqueous isotonic sterile injection solutions and aqueous and non-aqueous sterile suspensions known to be pharmaceutically acceptable carriers and well known to those of skill in the art may be employed for this purpose.

The therapeutic of the present invention may also be administered directly to the tumor in the form of a liquid, gel or suspension introduced by intratumoral injection. A study examining the treatment of mice bearing subcutaneous human pancreatic adenocarcinoma xenografts with recombinant Newcastle disease virus (rNDV) showed that intratumoral injection yielded better tumor regression than intravenous injection. In this study, animals were injected intratumorally every other day for a total of 4 injections, each containing 5×10⁷ 50% Tissue Culture Infective Dose (TCID50) rNDV in 50 μl (Buijs et al., 2015, Viruses, 6: 2980-2998). Similarly, another study examining the treatment of mice bearing subcutaneous bladder cancer xenografts with modified oncolytic adenovirus found that intratumoral injection significantly suppressed tumor growth. In this study, animals were injected intratumorally twice at a 1-day interval with 5×10⁸ infectious unit (IFU) viruses in 100 μl (Yang et al., 2015, Cell Death and Disease, e1760). Shown to be well-tolerated in Phase 1 trials, Pexa-Vec (JX-594), an oncolytic and immunotherapeutic vaccinia virus has been examined as an intratumorally-administered treatment for patients with advanced hepatocellular carcinoma (HCC). In this study design, patients were to be injected intratumorally 3 times every 2 weeks at one of 2 dose levels: 1×10⁸ plaque forming units (pfu), or 1×10⁹ pfu (Walther and Stein, 2015, Methods in Molecular Biology, 1317: 343-357).

A single administration of oncolytic adenoviral vectors of the invention may have therapeutic effects. However, in some embodiments of the invention, oncolytic adenoviral vectors or pharmaceutical compositions are administered several times during the treatment period. Oncolytic adenoviral vectors or pharmaceutical compositions may be administered for example from 1 to 10 times in the first 2 weeks, 4 weeks, monthly or during the treatment period. In one embodiment of the invention, administration is done three to seven times in the first 2 weeks, then at 4 weeks and then monthly, in a specific embodiment of the invention, administration is done four times in the first 2 weeks, then at 4 weeks and then monthly. The length of the treatment period may vary, and for example may last from two to 12 months or more.

To improve the efficacy of the present invention, in some embodiments, the therapeutic of the present invention is administered with an adjuvant. Suitable adjuvants include aluminum salts (alum) such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate, Incomplete Freund's Adjuvant (IFA), and monophosphoryl lipid A (MPL). These adjuvants are suitable for human administration, either alone or optionally all combinations thereof (Chang et al., “Adjuvant Activity of Incomplete Freund's Adjuvant,” Adv Drug Deliv Rev 32:173-186 (1998), which is hereby incorporated by reference in its entirety). Other adjuvants include cytokines, such as interleukins (IL-1, IL-2, and IL-12), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), di- and tri-palmitoyl-S-glyceryl cysteine (Pam₂Cys and Pam₃Cys, respectively), a TLR2 agonist, an anti-granulocyte macrophage colony-stimulating factor (GM-CSF) antibody, RR-XS15, Montanide®, and MALP-2.

The adenoviral vector of the present invention can be administered orally, parenterally, for example, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, or by application to mucous membranes, such as, that of the nose, throat, and bronchial tubes. They may be administered alone or with suitable pharmaceutical carriers, and can be in solid or liquid (e.g., aqueous) form such as, tablets, capsules, powders, solutions, suspensions, or emulsions.

The therapeutic of the present invention may be orally administered, for example, with an inert diluent, or with a suitable edible carrier, or they may be enclosed in hard or soft-shell capsules, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, the therapeutic may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like. Various other materials may be present as coatings or to modify the physical form of the dosage unit.

The therapeutic may also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols such as, propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

The therapeutic of the present invention may also be administered directly to the airways in the form of an aerosol. For use as aerosols, the compounds of the present invention in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The materials of the present invention also may be administered in a non-pressurized form such as in a nebulizer or atomizer.

The therapeutic of this invention may be administered in sufficient amounts to transfect the desired cells and provide sufficient levels of integration and expression of the replicating virus to provide a therapeutic benefit without undue adverse effects or with medically acceptable physiological effects which can be determined by those skilled in the medical arts.

Dosages of the therapeutic will depend primarily on factors, such as the condition being treated, the age, weight, and health of the patient, and may thus vary among patients. The dosage will be adjusted to balance the therapeutic benefit against any viral toxicity or side effects.

The present invention also relates to a method of enhancing the delivery to and distribution within a tumor mass of therapeutic proteins expressed from viruses. For example, an adenovirus as described herein, optionally in a pharmaceutical composition as described herein, can be injected into a tumor mass such that the virus infects and lyses one or more tumor cell.

Combination Therapy.

The viral immunotherapy of the invention is effective alone, but combination of multiple adenoviral immunotherapies, or one or more adenoviral immunotherapies with any other therapies, such as traditional therapy, may be more effective than either one alone. For example, each agent of the combination therapy may work independently in the tumor tissue, the adenoviral vectors may sensitize cells to chemotherapy or radiotherapy and/or chemotherapeutic agents may enhance the level of virus replication or effect the receptor status of the target cells. The agents of combination therapy may be administered simultaneously or sequentially.

In a preferred embodiment of the invention, the method or use further comprises administration of concurrent radiotherapy to a subject. In another preferred embodiment of the invention, the method or use further comprises administration of concurrent chemotherapy to a subject. As used herein “concurrent” refers to a therapy, which has been administered before, after or simultaneously with the gene therapy of the invention. The period for a concurrent therapy may vary from minutes to several weeks. In some embodiments, the concurrent therapy lasts for some hours.

Agents suitable for combination therapy include but are not limited to afatinib, all-trans retinoid acid, azacitidine, azathioprine, bleomycin, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, 5-fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, temozolomide, teniposide, tioguanine, valrubicin, vinblastine, vincristine, vindesine and vinorelbine.

In some embodiments, the method or use further comprises administration of verapamil or another calcium channel blocker to a subject. “Calcium channel blocker” refers to a class of drugs and natural substances which disrupt the conduction of calcium channels, and if may be selected from a group consisting of verapamil, dihydropyridines, gallopamil, diltiazem, mibefradil, bepridil, fluspirilene and fendiline.

In some embodiments, the method or use further comprises administration of autophagy inducing agents to a subject. Autophagy refers to a catabolic process involving the degradation of a cell's own components through the lysosomal machinery. “Autophagy inducing agents” refer to agents capable of inducing autophagy and may be selected from a group consisting of, but not limited to, mTOR inhibitors (e.g., temsirolimus, sirolimus, everolimus, and ridaforolimus), P13K inhibitors (e.g., wortmannin, lithium, tamoxifen, chloroquine, bafilomycin, and temozolomide. In a specific embodiment of the invention, the method further comprises administration of temozolomide to a subject. Temozolomide may be either oral or intravenous temozolomide.

In some embodiments, the method or use further comprises administration of chemotherapy or anti-CD20 therapy or other approaches for blocking of neutralizing antibodies. “Anti-CD20 therapy” refers to agents capable of killing CD20 positive cells, and may be selected from a group consisting of rituximab and other anti-CD20 monoclonal antibodies. “Approaches for blocking of neutralizing antibodies” refers to agents capable of inhibiting the generation of anti-viral antibodies that normally result from infection and may be selected from a group consisting of different chemotherapeutics, immunomodulatory substances, corticoids and other drugs. These substances may be selected from a group consisting of, but not limited to, cyclophosphamide, ciclosporin, azathioprine, methylprednisolone, etoposide, CD40L, CTLA4, FK506 (tacrolimus), IL-12, IFN-γ, interleukin 10, anti-CD8, anti-CD4 antibodies, hematopoietic stem cell transplantation (HSCT) and oral adenoviral proteins.

In some embodiments, the oncolytic adenoviral vector of the invention induces virion-mediated oncolysis of tumor cells and activates human immune response against tumor cells. In some embodiments, the method or use further comprises administration of substances capable to downregulating regulatory T-cells in a subject in an amount to downregulate (e.g., by at least 10%, 20%, 50%, 70%, 90% or more) regulatory T-cells in the subject. “Substances capable to downregulating regulatory T-cells” refers to agents that reduce the numbers of cells identified as T-suppressor or Regulatory T-cells. These cells have been identified as consisting one or many of the following immunophenotypic markers: CD4+, CD25+, FoxP3+, CD127− and GITR+. Such agents reducing T-suppressor or Regulatory T-cells may be selected from a group consisting of anti-CD25 antibodies or chemotherapeutics.

In some embodiments, the method or use further comprises administration of cyclophosphamide to a subject. Cyclophosphamide is a common chemotherapeutic agent, which has also been used in some autoimmune disorders. In the present invention, cyclophosphamide can be used to enhance viral replication and the effects of GM-CSF induced stimulation of NK and cytotoxic T-cells for enhanced immune response against the tumor. It can be used as intravenous bolus doses or low-dose oral metronomic administration.

Kits

The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration.

EXAMPLES

The present invention is further described by the following examples, which are illustrative of specific embodiments of the invention, and various uses thereof. These exemplifications, which illustrating certain specific aspects of the invention, do not portray the limitations or circumscribe the scope of the disclosed invention.

Unless otherwise indicated, the practice of the present invention employs conventional techniques of cell culture, molecular biology, microbiology, recombinant DNA manipulation, immunology science, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g. Cell Biology: a Laboratory Handbook: J. Cells (Ed). Academic Press. N.Y. (1996); Graham, F. L. and Prevec, L. Adenovirus-based expression vectors and recombinant vaccines. In: Vaccines: New Approaches to Immunological Problems. R. W. Ellis (ed) Butterworth. Pp 363-390; Grahan and Prevec Manipulation of adenovirus vectors. In: Methods in Molecular Biology, Vol. 7: Gene Transfer and Expression Techniques. E. J. Murray and J. M. Walker (eds) Humana Press Inc., Clifton, N.J. pp 109-128, 1991; Sambrook et al. (1989), Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press; Sambrook et al. (1989), and Ausubel et al. (1995), Short Protocols in Molecular Biology, John Wiley and Sons.

Example 1. Virology and Recombinant Nucleic Acids

Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, and microbial culture and transformation (e.g., electroporation, lipofection). Generally, enzymatic reactions and purification steps are performed according to the manufacturer's specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which are provided throughout this document. The nomenclature used herein and the laboratory procedures in analytical chemistry, organic synthetic chemistry, and pharmaceutical formulation and delivery, and treatment of patients. Methods for the construction of adenoviral mutants are generally known in the art. See Bett, A. J. et al, PNAS 1994 vol: 91, pages 8802-8806, Mittal, S. K., Virus Res., 1993, vol: 28, pages 67-90; and Hermiston, T. et al., Methods in Molecular Medicine: Adenovirus Methods and Protocols, W. S. M. Wold, ed, Humana Press, 1999. Further, the adenovirus 5 genome is registered as GenBank 10 accession #M73260, and the virus is available from the American Type Culture Collection, Rockville, Md., U.S.A., under accession number VR-5.

Viruses and Cell Lines

Adenoviruses and cell lines were generally obtained from American Type Culture Collection (ATCC), Manassas, Va.) unless otherwise noted. Cell lines used in the Examples below may include one or more of the cell lines listed in Table 2.

TABLE 2 Viruses and Cell Lines Cell Line Cell/Tissue Type Catalog # HEK-293 Normal human embryonic kidney ATCC ® CRL-1573 ™ MRC-5 Normal human lung ATCC ® CCL-171 ™ HMEC-1 Normal human endothelium ATCC ® CRL3243 ™ MCF 10A Normal human mammary epithelial ATCC ® CRL-10317 ™ NHLF Normal human lung fibroblasts Lonza CC-2512 HUVEC Normal human umbilical endothelium ATCC ® CRL-1730 ™ HCC827 lung adenocarcinoma ATCC ® CRL-2868 ™ A549 human lung carcinoma ATCC ® CCL-185 ™ ADS-12 a derivative from a murine KRAS mutant lung adenocarcinoma cell line (LKR-13) NCI-H1734 NSCLC adenocarcinoma ATCC ® CRL-5891 ™ NCI-H2110 NSCLC metastatic carcinoma ATCC ® CRL-5924 ™ HT-1080 Connective tissue fibrosarcoma ATCC ® CCL121 ™ PC-3 Prostate adenocarcinoma ATCC ® CRL-1435 ™ , SNU-449 Hepatocellular carcinoma ATCC ® CRL-2234 ™ HepG2 Hepatocellular carcinoma ATCC ® HB-8065 ™ MDA-MB-231 Breast adenocarcinoma ATCC ® HTB-26 ™ PANC-1 Pancreatic carcinoma ATCC ® CRL-1469 ™ SW780 Bladder carcinoma ATCC ® CRL-2169 ™ FaDu Head and neck squamous cell carcinoma ATCC ® HTB43 ™ DLD-1 Colorectal adenocarcinoma ATCC ® CCL-221 ™ CT26 Murine colon carcinoma ATCC ® CRL-2638 ™ U-87 brain glioblastoma ATCC ® HTB14 ™ MBT-2 Murine bladder cancer described, e.g., in Takahashi et al., J Urol, 166(6), 2506-2511 B16F10 Murine skin melanoma ATCC ® CRL-6475 ™ TAV-255 D19 Adenovirus described in, e.g., Zhang et al., Cancer Gene Ther. (2015) 22(1):17-22. Ad5 Adenoid 75 strain ATCC ® VR-5 ™

Viral Purification and Quantitation

Viral stocks were propagated on HEK-293 cells and purified by standard methods such as column purification kits (Virapure, Millipore) or CsCl gradient centrifugation (as described in Tollefson, A., Hermiston, T. W., and Wold, W. S. M.; “Preparation and Titration of CsCl-banded Adenovirus Stock” in Adenovirus Methods and Protocols, Humana Press, 1999, pp 1-10, W. S. M. Wold, Ed.). The method used to quantitate viral particles is based on simple OD 260/280 readings, e.g., using the method of Lehmberg et al. (1999) J. Chrom. B, 732:411-423. In the viral concentration range used, the A260 nm peak area of each sample is directly proportional to the number of viral particles in the sample. The number of viral particles per ml in each test sample was calculated by multiplying the known number of viral particles per ml in the standard by the ratio of the A260 nm viral peak area of the sample to the A260 nm viral peak area of the standard. One A260 unit contains approximately 1×10¹² viral particles. Virus Infectious Units/ml (IU/ml) were determined by hexon staining of infected cells (e.g., using Adeno-X™ Rapid titer kit from Takara Bio USA, Inc. (TBUSA, formerly known as Clontech Laboratories, Inc.).

Example 2. Bioselection

Initial screening of recombinant virus was based on isolated viral DNA rescued from transfected 293 cells where viral propagation based cytopathic effects (CPE) were observed. Viral DNA was used as a template for PCR based detection of sequences flanking the site of the TAV-255 E1a enhancer deletion region. Wild-type sequence would produce a band of 350 bp, while DNA which had the E1a enhancer deletion would only generate a clearly distinguishable 300 bp band. In addition, primers specific for internal sequences of the IL-12 transgene and for Ad hexon sequences were used to verify 400 bp and 3 kb PCR amplified bands respectively that would only be present in viral DNA generated by recombination between the two parental plasmids containing either the E1 region and transgene insert or the late Ad structural proteins derived solely from the pAdEasy™ plasmid. Individual constructs were isolated by two rounds of plaque purification on A549 or 293 cells using standard methods (Tollefson, A., Hermiston, T. W., and Wold, W. S. M.; “Preparation and Titration of CsCl-banded Adenovirus Stock” in Adenovirus Methods and Protocols, Humana Press, 1999, pp 1-10, W. S. M. Wold, Ed). Dilutions of adenoviral lysates were used to infect A549 or 293 cells in a standard plaque assay. Well-individuated plaques were harvested, and the same plaque assay method was used to generate a second round of individual plaques from these harvests. Well isolated plaques from the second round of plaque purification were deemed pure, infected cultures are prepared using these purified plaques, and the oncolytic potency and selectivity of these culture supernatants was determined.

Example 3. Cytolytic Assay

Tumor specific viral lysis was evaluated in both tumor and non-tumor cell lines of murine and human origin by infection of the cells in vitro with the viruses, followed by standard crystal violet for cell viability over time as instructed in the kit manuals.

Example 4. DNA Sequencing

DNA sequencing of the human Ad5-based recombinant adenovirus genomic DNAs was performed as follows. Viral DNA was purified from recombinant adenoviruses such as TAV-255 or other modifications of these constructs by standard column purification methods such as the Qia-amp® blood DNA purification kit from Qiagen®. PCR primers were used to amplify and isolate regions covering the E1a modified regions and the regions containing the transgene insert and sent out for sequencing at a CRO. Isolated DNA was also analyzed by standard restriction digestion and SDS PAGE analysis for verification of appropriate sized bands of digested DNA. Sequence information was analyzed using the Vector NTI program (Informatix).

Example 5. Construction of Recombinant Viruses

The base shuttle transfer vector includes pXC1 TAV 255 d19k (Zhang et al., Cancer Gene Ther. (2015) 22(1):17-22). This plasmid has a deletion between bp −305 to −255 of the E1a enhancer region, removing two Pea3 and one E2F binding sites which restrict replication and oncolysis of an adenovirus with this deletion to infected tumor cells (Hedrun, F. H., Shantanu K., and Reid, T. (2011) Cancer Gene Therapy 18; 717-723.) It also contains sites allowing deletion of the E1b 19k region and exogenous transgene insert by a Sal I/Xho I digest. Without the digest, the majority of the E1b 19k region is intact, but non-functional. cDNAs for each transgene of interest were synthesized (GeneArt™) or isolated from commercial plasmid sources (GE Lifesciences or GeneCopoeia™) by PCR using primers which added on 5′ SalI and 3′ XhoI restriction sequences for insertion into the SalI/XhoI digested pXC1 TAV 255 plasmids. The modified pXC1 TAV 255 gene insert plasmids were amplified in E. coli and purified using Qiagen® Maxi-prep plasmid kits.

To obtain recombinant adenoviruses, the pXC1 TAV 255 gene insert-containing plasmids were co-transfected into HEK-293 cells (ATCC) with pBHG10 (Microbix) as described (Bett, A. J., Haddara, W., Prevec, L., and Graham, F. (1994) PNAS 91; 8802-8806), using the calcium phosphate transfection protocol from Molecular Cloning: A laboratory manual (Maniatis Vol. 3; 16.30-16.36) for 2-5 μg plasmid DNA (for both plasmids so the pXC1 will be in molar excess) per 60 mm dish of cells. Recombination between homologous adenovirus sequences from each plasmid generates a full length, replication competent adenovirus containing the E1 modifications described earlier and a specific transgene inserted into the E1b 19k deletion site and the E3 deletion supplied by the pBHG10 plasmid. Optionally, the pBHG10 is provided as the adenoviral genome source, having substantial additional utility over vectors such as pJM17 (See, e.g., Hedrun et al, (2011)). Preferentially, an E3 deletion and/or other modifications allow increased packaging capacity for exogenous genes in excess of pJM17 capacity.

In another embodiment, the human IL-12 virus, TRZ627, was constructed using a modification of the pAdEasy™ Adenoviral Vector System (Agilent Technologies). First, sequences from pXCI-TAV d19K plasmid (Hedjran F et al., Cancer Gene Therapy (2011) 18, 717-723) which included the 50-nucleotide deletion in the enhancer of E1A which restricts viral propagation to tumor vs. non-tumor cells, were subcloned into the pShuttle™ vector supplied in the pAdEasy™ kit to create the TAV-255 Shuttle E1 cloning plasmid. Sequences between the first Pac I site (6) and the single Mfe I site (807) in pShuttle were replaced by pXC1 TAV d19K sequences from the beginning of the 5′ ITR sequence (21) to the Mfe I site (3874), corresponding to the same Mfe I site in pShuttle. A Pac I cloning site was added by PCR onto the 5′ end of this fragment, which brought in Ad E1a and E1b sequence not found in the original pShuttle plasmid. The TAV-255 deletion of 50 bp in the E1a enhancer region removes the Pea3 III, Pea3 II, and an E2F transcription factor binding sites and corresponds to human wild-type Ad5 sequence of bases 194-244. The added sequences also included a modification introducing Sal I/Xho I cloning sites into the E1b 19k coding region which can be used to replace the E1b 19k ORF with an exogenous transgene insert whose expression would be driven by the Ad E1b promoter during viral replication. Recombination between Pme I linearized pTAV-255 Shuttle E1 containing hIL-12 cloned in at the E1b 19k site and the pAdEasy vector took place in the recA proficient BJ5183 bacterial strain, which had been modified to already contain the pAdEasy plasmid. DNA isolated from Kanamycin resistant plated colonies was screened for full-length viral DNA recombinants by restriction digest. Positive clones (TRZ-627, hIL-12) were subsequently digested with Pac I to free up the Ad ITRs and then transfected into 293 cells to amplify the virus.

Infected HEK-293 cells were collected when visible sign of cytopathic effects due to viral replication were observed up to 2 weeks post-infection and resuspended in their media and lysed by 3 rounds of freeze/thaw. Virus can be purified from the lysate by several methods, including Anion-exchange HPLC (Shabram, P. W., et al (1997) Human Gene Therapy 8; 453-465) or several commercially available kits based on affinity chromatography or size exclusion membranes or columns (e.g., Adeno-X™ Maxi Purification Kit, Clontech® (Takara), Adenovirus Purification Virakit®, Virapur®). Purified virus can also undergo clonal isolation by standard plaque purification methods, followed by re-amplification and purification of the plaque purified viral clone.

Example 6. In Vivo Demonstration of Reduction in Tumor Volume with Oncolytic Adenoviral Vectors Encoding Immunomodulatory Polypeptides: Single Viruses with and without Antibody Treatment Materials and Methods

An anti-mPD-L1 antibody is, e.g., from BioXCell®, Catalog #BE0101 (Rat IgG2b). This antibody was used in the below experiments.

Virus samples were stored in 25 mM NaCl, 10 mM Tris Tris(hydroxymethyl)aminomethane), and 5% glycerol with a pH value of 8.0. Vials were stored protected from light at −80° C. On each day of dosing, one vial was thawed at room temperature for approximately 20 minutes. A single dose is 1×10⁹ pfu.

The mouse tumor model in this Example uses syngeneic immunocompetent mice. Female Jackson 129S1 (129S1/Sv1mJ) mice were used in this study. They were 6-7 weeks old on Day 1 of the experiment. The animals were fed irradiated Harlan 2918.15 Rodent Diet and water ad libitum. Animals were housed in static cages with Bed-O'Cobs® bedding inside bioBubble® Clean Rooms that provide H.E.P.A filtered air into the bubble environment at 100 complete air changes per hour. All treatments, body weight determinations, and tumor measurements were carried out in the bubble environment. The environment was controlled to a temperature range of 70°±2° F. and a humidity range of 30-70%.

Cell Preparation

ADS-12 cells were grown in RPMI 1640 medium which was modified with 1% 100 mM Na pyruvate, 1% 200 mM L-glutamine, 1% 1M HEPES buffer, 1% of a 45% glucose solution and supplemented with 10% non-heat-inactivated Fetal Bovine Serum (FBS) and 1% 100× Penicillin/Streptomycin/L-Glutamine (PSG). The growth environment was maintained in an incubator with a 5% CO₂ atmosphere at 37° C. When expansion was complete, the cells (passage 7) were trypsinized using 0.25% trypsin/2.21 mM EDTA in HBSS solution. Following cell detachment, the trypsin was inactivated by dilution with complete growth medium and any clumps of cells were separated by pipetting. The cells were centrifuged at 200 rcf for 8 minutes at 4° C., the supernatant was aspirated, and the pellet was re-suspended in cold Dulbecco's Phosphate Buffered Saline (DPBS) by pipetting. An aliquot of the homogeneous cell suspension was diluted in a trypan blue solution and counted using a Luna automated cell counter. The cell suspension was centrifuged at 200 rcf for 8 minutes at 4° C. The supernatant was aspirated and the cell pellet was re-suspended in cold Dulbecco's Phosphate Buffered Saline (DPBS) to generate a final concentration of 1×10⁷ trypan-excluding cells/ml. The cell suspension was maintained on wet ice during implantation. Following implantation, an aliquot of the remaining cells was diluted with a trypan blue solution and counted to determine the post-implantation cell viability. The cell viabilities of the suspensions used for implantation (two preps) are listed in Table 3.

TABLE 3 Implantation Cell Viability Pre-Implant Viability (%) Post-Implant Viability (%) Cell Prep 1 95 89 Cell Prep 2 95 89

Test animals were implanted subcutaneously on both flanks (on the back between the spine and the hip), the right flank on Day 0 and the left flank on Day 8, with 1×10⁶ cells in 0.1 ml of serum-free medium using a 28-gauge insulin syringe with a fixed needle.

All mice were sorted into study groups based on caliper measurement estimation of tumor burden on Day 15 when the mean tumor burden for all animals on the right flank was approximately 82 mm³ (range of group means, 75-90 mm³). The mice were distributed to ensure that the mean tumor burden on the right flank for all groups was within 10% of the overall mean tumor burden for the study population.

Results

The mean estimated right side tumor burden for all groups in the experiment on the first day of treatment was 82 mm3 and all of the groups in the experiment were well-matched (range of group means, 75-90 mm3). All animals weighed at least 13.3 g at the initiation of therapy. Mean group body weights at first treatment were also well-matched (range, 15.4-18.3 g). A tumor burden of 500 mm3 was chosen for evaluation of efficacy by tumor growth delay for the right and left tumors. The median Control Group (FIG. 6A) tumor burdens reached 500 mm3 on Day 47 for right tumors and Day 43 for left tumors. The median tumor volume doubling times for the Control Group were 12.1 and 10.1 days for the right and left tumors, respectively. Control animals experienced a 7.3% mean weight gain during the treatment regimen. There were no spontaneous regressions in the Control Group for either right or left side tumors, however, 50% of the tumors on the left side never reached the palpation limit. In some embodiments, mice with palpable left side tumors were put on study. Since in this experiment the implant was delayed 8 days, the left side tumors were not palpable at staging. These mice that had a left side tumor that remained a 0 throughout the study could have been triaged out of the study if implanted on the same day as the right side.

Results of mouse inoculation and tumor growth are shown in FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG. 6F, FIG. 6G, FIG. 6H, FIG. 6I, FIG. 6J, FIG. 6K, and FIG. 6L. The series of graphs shows treatment of tumor bearing mice (n=8 in each group) with oncolytic viruses comprising various transgenes, with or without anti-PD-L1.

FIG. 6A is a graph showing the activity of various oncolytic viruses compared to the empty virus (“d19k”), 38 days after cell implantation (primary tumor) as a function of tumor growth inhibition. Black bars represent virus alone and hatched bars represent virus+anti-PD-L1 antibody. FIG. 6B is a graph comparing the average tumor size (primary tumor) over time of tumors injected with virus buffer (black circles), empty vector (blue solid squares), anti-PD-L1 alone (antibody given on days 16, 20, 24, and 28, right-hand arrow in each pair of arrows), each of five viruses alone (viruses carrying CTLA-4, IL-12, IL-7, CD70, and IL-10 transgenes) or combined with anti-PD-L1 antibody. FIG. 6C shows similar data for three more viruses (viruses carrying OX40L, CD40L, and GM-CSF transgenes). As shown in FIG. 6A, FIG. 6B, and FIG. 6C, the IL-12 oncolytic virus treatment, alone or in combination with an anti-PD-L1 antibody, were the most effective at reducing tumor growth. CD70, IL-7, and CTLA-4 viruses were also able to reduce tumor volume significantly when combined with the anti-PD-L1 antibody.

The following Figures (FIG. 6D, FIG. 6E, FIG. 6F, FIG. 6G, FIG. 6H, FIG. 6I, FIG. 6J, FIG. 6K, and FIG. 6L) demonstrate efficacy (or lack thereof) of various viruses with transgenes with or without PD-L1 on the primary tumor only; the thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and the anti-PD-L1 antibody, if used (intraperitoneal, right arrows). Treatments are shown for the primary tumor (receiving the oncolytic virus injection) and are as follows: FIG. 6D, virus buffer and control IgG only (left) and virus buffer and anti-PD-L1 antibody (right); FIG. 6E, d19k (empty virus) and control IgG only (left) and d19k and anti-PD-L1 antibody (right); FIG. 6F, CTLA-4 virus with control IgG (left) or anti-PD-L1 (right); FIG. 6G, IL-12 virus with control IgG (left) or anti-PD-L1 antibody (right); FIG. 6H, GM-CSF virus with control IgG (left) or anti-PD-L1 antibody (right); FIG. 6I, IL-7 virus with control IgG (left) or anti-PD-L1 antibody (right); FIG. 6J, CD40L virus with control IgG (left) or anti-PD-L1 antibody (right); FIG. 6K, L10 trap virus with control IgG (left) or anti-PD-L1 antibody (right); and FIG. 6L, OX40L virus with control IgG (left) or anti-PD-L1 antibody.

As shown in FIG. 6G, FIG. 6I, FIG. 6F and FIG. 6J, combinations with the IL-12 adenovirus (FIG. 6G), showed the greatest ability to reduce tumor volume. As in FIG. 6A, the IL-7 (FIG. 6I) and CTLA-4 (FIG. 6F) oncolytic viruses also showed activity when combined with anti-PD-L1 antibody. Treatment with the oncolytic virus encoding the CD40 ligand also showed some activity when combined with the anti-PD-L1 antibody (FIG. 6J).

Example 7. In Vivo Demonstration of Reduction in Tumor Volume with Oncolytic Adenoviral Vectors Encoding Immunomodulatory Polypeptides: Virus Mixing Study

The mouse tumor model in this Example uses syngeneic immunocompetent mice. Animals were injected with approximately 1,000,000 cells subcutaneously (s.c.) into the right hind flank of the mouse. At the same time, the mice were also injected with 1,000,000 cells s.c. into the left hind flank of the mouse to create a bilateral tumor model. When the right (primary) tumors reached 63-80 mm³ in size and the left tumor was palpable, they were injected with 25 μl of virus buffer or 25 μl of virus at 4×10⁹ pfu/ml (plaque forming units per ml) directly into the center of the primary tumor every fourth day for a total of three doses. The mice were also treated intraperitoneally with 250 μl of an anti-PD-L1 antibody at 2 mg/ml every fourth day for a total of three doses. The antibody was administered 24 hours after administration of the virus. A reduction in the size of the primary and distal (contralateral) tumor would be noted relative to the virus buffer control and additional controls such as the wild-type virus (not expressing a transgene) with or without administration of the anti-PD-L1 antibody. A specific example of the treatment with oncolytic adenoviral vectors is described in more detail below for a panel of such vectors.

Evaluation of the primary tumor (right flank) was used to determine the direct effect of the oncolytic viruses whereas evaluation of the contralateral tumor (left flank) was used to see the systemic effects of the oncolytic viruses. ADS-12 (a murine KRAS-mutant lung adenocarcinoma cell line) grown in its syngeneic mouse strain is a tumor model known to support adenoviral infection and replication and is useful in the evaluation of host immune responses to oncolytic human adenoviruses.

Materials and Methods

An anti-mPD-L1 antibody is, e.g., from BioXCell®, Catalog #BE0101 (Rat IgG2b). This antibody was used in the below experiments.

Virus samples were stored in 25 mM NaCl, 10 mM Tris Tris(hydroxymethyl)aminomethane), and 5% glycerol with a pH value of 8.0. Vials were stored protected from light at −80° C. On each day of dosing, one vial was thawed at room temperature for approximately 20 minutes.

Animal Studies

Female 129S1 (129S1/SvImJ) mice from The Jackson Laboratory were used in this study. They were approximately 7-8 weeks old on Day 14 of the experiment. The animals were fed irradiated Harlan 2918.15 Rodent Diet and water ad libitum. Animals were housed in static cages with Bed-O'Cobs® bedding inside BioBubble® Clean Rooms that provide H.E.P.A filtered air into the bubble environment at 100 complete air changes per hour. All treatments, body weight determinations, and tumor measurements were carried out in the bubble environment. The environment was controlled to a temperature range of 70°±2° F. and a humidity range of 30-70%. All procedures carried out in this experiment were conducted in compliance with all the laws, regulations and guidelines of the National Institutes of Health (NIH) and with the approval of Molecular Imaging, Inc.'s Animal Care and Use Committee. Molecular Imaging, Inc. is an AAALAC accredited facility.

Cell Preparation

ADS-12 cells (murine KRAS-mutant lung adenocarcinoma) were grown in RPMI 1640 medium which is modified with 1% 100 mM Na pyruvate, 1% 200 mM L-glutamine, 1% 1M HEPES buffer, 1% of a 45% glucose solution and supplemented with 10% non-heat-inactivated Fetal Bovine Serum (FBS) and 1% 100× Penicillin/Streptomycin/L-Glutamine (PSG). The growth environment was maintained in an incubator with a 5% CO₂ atmosphere at 37° C. When expansion as complete, the cells are trypsinized using 0.25% trypsin/2.21 mM EDTA in HBSS solution. Following the cell viabilities of the suspensions used for implantation (two preps) are listed in the table below.

TABLE 4 Implantation Cell Viability Pre-Implant Viability (%) Post-Implant Viability (%) Cell Prep 1 94 92 Cell Prep 2 97 92

Test animals were implanted subcutaneously, on both flanks (on the back between the spine and the hip) on Day 0 with 1.00×10⁶ cells in 0.1 ml of serum-free medium using a 28-gauge insulin syringe with a fixed needle.

Treatment

All mice were sorted into study groups based on caliper measurement estimation of tumor burden on Day 14 when the mean tumor burden for all animals on the right flank is approximately 68 mm³ (range of group means, 65-71 mm³). The mice were distributed to ensure that the mean tumor burden for all groups was within 10% of the overall mean tumor burden for the study population.

Measurement and Endpoints

Tumor burden (mm³) was estimated from caliper measurements by the formula for the volume of a prolate ellipsoid assuming unit density as: Tumor burden (mm³)=(L×W²)/2, where L and W are the respective orthogonal tumor length and width measurements (mm). All groups were compared to the virus buffer control group.

The primary endpoints used to evaluate efficacy were: tumor growth delay, complete and partial tumor response, and the number of tumor-free survivors at the end of the study for both left and right tumors. A complete response (CR) is defined as a decrease in tumor mass to an undetectable size (<63 mm³), and a partial response (PR) is defined as a smaller tumor mass at the last measurement compared to at the first treatment. PRs are exclusive of CRs.

All animals were observed for clinical signs at least once daily. Animals were weighed on each day of treatment. Individual body weights were recorded three times weekly. Animals with combined tumor burdens in excess of 2000 mm³ were euthanized, as were those found in obvious distress or in a moribund condition. Treatment-related weight loss in excess of 20% is generally considered unacceptably toxic. In this Example, a dosage level was determined to be tolerated if treatment-related weight loss (during and two weeks after treatment) was <20% and mortality during this period in the absence of potentially lethal tumor burdens was ≤10%.

Results

Results of mouse inoculation and tumor growth are shown in FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, FIG. 7F, FIG. 7G, FIG. 7H, FIG. 7I, FIG. 7J, FIG. 7K, FIG. 7L, FIG. 7M, FIG. 7N, FIG. 7O, FIG. 7P, FIG. 7Q, FIG. 7R, FIG. 7S, FIG. 7T, FIG. 7U, FIG. 7V, FIG. 7W, FIG. 7X, FIG. 7Y, FIG. 7Z, and FIG. 7AA. The series of graphs shows treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising various transgenes, with or without anti-PD-L1. The left-hand panel of each Figure represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and the anti-PD-L1 antibody, if used (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). Treatments shown are as follows: FIG. 7A, virus buffer only; FIG. 7B, empty virus only (TRZ000); FIG. 7C, TRZ010 (IL-10trap)+empty virus; FIG. 7D, TRZ011 (OX40 ligand)+empty virus; FIG. 7E, TRZ009 (CD70)+empty virus; FIG. 7F, TRZ007 (IL-7)+empty virus, FIG. 7G, TRZ002 (IL-12)+empty virus; FIG. 7H, TRZ004 (GM-CSF)+empty virus; FIG. 7I, TRZ003 (flagellin)+empty virus; FIG. 7J, TRZ002+TRZ010; FIG. 7K, TRZ002+TRZ007; FIG. 7L, TRZ007+TRZ010; FIG. 7M, TRZ011+TRZ004; FIG. 7N, TRZ009+TRZ003; FIG. 7O, TRZ002+TRZ009; FIG. 7P, TRZ007+TRZ009; FIG. 7Q, TRZ007+TRZ004; FIG. 7R, TRZ002+TRZ011; FIG. 7S, TRZ010+TRZ004; FIG. 7T, TRZ002+TRZ004; FIG. 7U, virus buffer and anti-PD-L1; FIG. 7V, TRZ002+empty virus+anti-PD-L1; FIG. 7W, TRZ009+anti-PD-L1; FIG. 7X, TRZ007+empty virus+anti-PD-L1; FIG. 7Y, TRZ002+TRZ007+anti-PD-L1; FIG. 7Z, TRZ007+TRZ009+anti-PD-L1; and FIG. 7AA, TRZ002+TRZ009+anti-PD-L1. All viruses were administered at 1×10⁸pfu/dose for each virus, resulting in 2×10⁸ pfu total in virus combinations.

As can be seen in FIG. 7G, FIG. 7K, FIG. 7V and FIG. 7AA, certain combinations of virus transgenes were extremely successful in lysing tumor cells, including a number of examples of complete response. For example, FIG. 7G shows tumor volume in mice treated with an IL-12 adenovirus. 5 out of 8 mice in FIG. 7G had a complete response, and one mouse had a partial response. In the contralateral tumor, however, no complete or partial responses were seen, indicating that there were no systemic immune effects from treatment with the IL-12 virus alone. A better result is seen in FIG. 7K, which shows mice treated with the same IL-12 adenovirus in combination with an IL-7 adenovirus. In this group of mice, 7 out of 8 had a complete response in the primary tumor (into which the virus was injected) and in the contralateral tumor two mice showed a complete response and one a partial response. In FIG. 7V (IL-12 adenovirus+anti-PD-L1 antibody) and FIG. 7AA (IL-12 adenovirus+CD70 adenovirus+anti-PD-L1 antibody), each group of 8 mice had 8 complete responses in the primary tumor and several in the contralateral tumor. These data demonstrate that certain combination therapies comprising oncolytic adenoviruses can be useful in treating primary and metastatic tumors.

Example 8. Adenoviral Vectors Expressing Multiple Immunomodulatory Polypeptides

If deletions in the adenovirus backbone are sufficiently large enough to allow packaging of viral DNA containing two exogenously added transgenes into the viral capsids, the two genes can be co-expressed by several methods from a single deletion site in adenovirus. Both added genes can be linked to each other by methods described below and have their expression controlled by an endogenous adenovirus promoter, not an exogenously added promoter, so that high expression will only occur during conditions of viral replication. Control of restricting viral replication to certain conditions such as after infection of tumor cells, is described elsewhere in this document. Co-expression of two proteins from a single transcript can be achieved through the use of virus components such as internal ribosome entry site (IRES) elements (Renaud-Gabardos E et al, World J Exp Med 2015, 5: 11-20), insertion of self-cleaving 2A peptide sequences derived from viruses such as Foot and Mouth Disease virus (FMDV) (Garry A. Luke (2012), Translating 2A Research into Practice, Innovations in Biotechnology, Dr. Eddy C. Agbo (Ed.), ISBN: 978-953-51-0096-6, InTech), or by combining the sequences of the two transgenes into a single fusion protein. An exemplary method would use one of the 2A sequences to direct more equal level of expression from both transgenes as opposed to lower expression levels typically seen from the second transgene when using IRES elements.

To maintain co-expression from two single gene inserts at different sites, the cDNA for each transgene can be inserted into separate deleted regions of adenovirus so that expression of each would be controlled separately by the endogenous upstream adenovirus promoter. No exogenous promoter would be added with the exogenous transgene sequence. Expression of the E1a proteins leads to the activation of the other adenovirus promoters and viral replication, so expression from each endogenous adenovirus promoter is linked to viral replication. Exogenous transgenes inserted behind different adenovirus promoters, such as the E1b promoter, the E3 promoter, and the E4 promoter, in place of deletions in these regions, leads to a construct where co-expression from each inserted transgene is limited to conditions of where viral replication occurs. Combined with modifications in the E1a enhancer region as described previously in this document to restrict viral replication to tumor cells, co-expression of both exogenous transgenes is restricted to tumor cells.

As shown schematically in FIG. 2, provided is an ΔE1b19K site that includes a deletion of bp 1714-1916 (numbered according to hAd5 vector sequence), which increases packaging capacity by approximately 200 bp. Also provided is a deletion at the E3 site (ΔE3 site as shown in FIG. 2); this deletion can be any deletion in the E3 region, generally at or about bp 27,900-30,800 bp (numbered according to hAd5 vector sequence), which increases packaging capacity up to an additional approximately 2400 bp, as compared to most wild type adenoviruses, which are typically limited to containing (i.e., packaging) approximately 1800 bp of exogenous sequences.

Provided herein are deletions in E3 open reading frames that are suitable for modification (e.g., truncation or deletion) without substantially decreasing viral propagation. By way of non-limiting example, provided are adenoviral vectors wherein the E3 12.5K coding region (27,852-28,175 bp) is truncated (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 320 or greater than 320 bases are deleted from one or more truncation sites within the region) or entirely deleted. Also provided are adenoviral vectors wherein the E3 7.1K coding region (28,541-28,732) is truncated (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or greater than 190 bases are deleted from one or more truncation sites within the region) or entirely deleted. The 7.1K sequence is associated with inhibition of TRAIL apoptosis and associated with one or more RID proteins. Also provided are adenoviral vectors wherein the E3 gp19K (28,729-29211) is truncated (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450 or greater than 450 bases are deleted from one or more truncation sites within the region) or entirely deleted. The gp19K sequence is associated with inhibition of CTL killing. Also provided are adenoviral vectors wherein the E3 10.5 (also called E3 11.6 (ADP)) (29,485-29,766) is truncated (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, or greater than 250 bases are deleted from one or more truncation sites within the region) or entirely deleted. The 10.5 sequence is associated with promotion of virus release. Also provided are adenoviral vectors wherein the E3 (RIDα) (29,778-29,969) and/or the E3 (RIDβ) (30,057-30,455) is truncated (e.g., with respect to E3 (RIDα) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or greater than 190 bases are deleted from one or more truncation sites within the region) or entirely deleted, or truncated (e.g., with respect to E3 (RIDβ) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 390, or greater than 390 bases are deleted from one or more truncation sites within the region) or entirely deleted. These RID sequences are associated with inhibition of TNF, FasL, and TRAIL apoptosis and degrade EGFR. Any combination of the above-referenced deletions is also provided.

Also provided are adenoviral vectors wherein the E3 14.7K (30,488-30,834) is truncated (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 340, or greater than 340 bases are deleted from one or more truncation sites within the region) or entirely deleted. The 14.7K sequence is associated with inhibition of TNF, FasL, and TRAIL apoptosis.

Descriptions of exemplary dual transgene constructs shown in Table 5 in this Example and Table 6 in Example 13. As can be seen in the table, any of the transgenes may be inserted into the E1 or E3 region. For example, in one embodiment a dual transgene construct may have IL-12 inserted into the E1 region and IL-2 in the E3 region. In another embodiment, a dual transgene construct may have IL-2 inserted into the E1 region and IL-12 in the E3 region. Use of the dual transgene constructs in therapeutic oncolytic adenoviruses is described in Examples 9 and 13.

TABLE 5 Exemplary dual transgene constructs. TRZ Number Brief Description TRZ402* TAV-255-d19kE1-mIL-10T-E3-mIL-12 TRZ403* TAV-255-d19kE1-mIL-7-E3-mIL-12 TRZ404* TAV-255-d19kE1-mCD70-E3-IL-12 TRZ405 TAV-255-d19kE1-mOX40L-E3-mGM-CSF TRZ406 TAV-255-d19kE1-mIL-10T-E3-mIL-7 TRZ407 TAV-255-d19kE1-mIL-12-E3-mIL-10T TRZ408 TAV-255-d19k-E3-mIL-12 TRZ409 E1-mIL-12/E3-mIL-7 TRZ413 E1-mIL-7/E3-mIL-12-P2A-ADP TRZ418* E1-trimeric mCD70/E3-mIL-12 TRZ421* E1-mIL-2/E3-mIL-12 TRZ501 TAV-255-d19K Dual mIL-10T-P2A-mIL-12, E3 deleted TRZ510 E1-mIL-7-P2A-mIL-12 TRZ512 E1-mIL-12-P2A-mIL-7 m = murine; P2A = cleavage site *See also Table 6

Example 9. Adenoviral Vectors Expressing an Immunomodulatory Polypeptide from the E4 Region

A nucleic acid sequence encoding one or more immunomodulatory polypeptides is inserted into the adenoviral genome by truncation or deletion of a portion of the E4 region of the viral genome. An E4 deletion and exogenous insertion can be utilized in combination with any other viral modification provided herein or otherwise known in the art, or alternatively, without any other viral modification. Exemplary E4 regions useful as insertion sites include truncation or deletion of E4 ORF1 (35,136-35,522 bp) and/or E4 ORF2 (34,696-35,106 bp) (each numbered according to hAd5 vector sequence). The expression of the one or more immunomodulatory polypeptides is controlled by the endogenous E4 promoter. A schematic illustration of this embodiment is provided in FIG. 3.

Example 10. Adenoviral Vectors Comprising Altered Configuration of Pea3 Sites

Adenoviral vectors are designed in which sequences in and around Pea3 sites I-V near the E1a Enhancer region of adenoviral vectors, in which sequences in and around Pea3 sites are altered, moved, or deleted in order to produce vectors with a variety of expression characteristics. In one embodiment, adenovirus vectors are engineered which have lower affinity Pea3 sites compared to wild-type adenoviral vectors. Such vectors are designed to be efficient in cells in the tumor microenvironment where the concentration of transcription factors is high, but to be relatively inactive in normal (e.g., non-neoplastic) cells, thus reducing the possibility of side effects caused by damage to normal tissue cells. An illustration of the E1a area of wild-type and TAV-255 constructs is shown in FIG. 9A. A cartoon illustration of the proposed method of action of the E1a enhancer region mutants is shown in FIG. 9B. Constructs NV1 to NV7 are based in part on the TAV-255 construct.

In one embodiment, a vector is provided wherein, (in relation to the wild-type sequence) the sequence is removed between Pea3 IV and Pea3 III sites, a single mutation is made in Pea3 III, the sequence between Pea3 III and Pea3 II, and the Pea3 II site is mutated. The sequence of the E1a enhancer region of the exemplary construct NV1 is set forth in SEQ ID NO:99.

In another embodiment, a vector is provided wherein, (in relation to the wild-type sequence) the sequence is removed between Pea3 IV and Pea3 III sites, both Pea3 III and Pea3 II are mutated, and Pea3 V flanking sites are mutated such that the resultant Pea3 V site has an affinity more similar to Pea3 III and enhancer 1. The sequence of the E1a enhancer region of the exemplary construct NV2 is set forth in SEQ ID NO:100.

In another embodiment, a vector is provided wherein, (in relation to the wild-type sequence) the sequence between Pea3 V and Pea3 IV sites is replaced with the sequence between the Pea3 III and Pea3 II sites; the sequence between the Pea3 IV and Pea3 III is deleted; the sequence between the Pea3 III and Pea3 II is deleted; and the Pea3 III and Pea3 II sites are both mutated, as well as the residues 3 bp that are immediately 5′ of the Pea3 V site. The sequence of the E1a enhancer region of the exemplary construct NV3 is set forth in SEQ ID NO:101.

In another embodiment, a vector is provided wherein, (in relation to the wild-type sequence) the flanking sequences around the Pea3 III and Pea3 II sites are altered to mimic the lower affinity Pea3 V and Pea3 IV sites (thus engineering a vector in which all Pea3 sites except Pea3 I are lower affinity). The sequence of the E1a enhancer region of the exemplary construct NV4 is set forth in SEQ ID NO:102.

In another embodiment, a vector is provided wherein, (in relation to the wild-type sequence) the flanking sequences around the Pea3 III and Pea3 II sites are altered to mimic the lower affinity Pea3 V and Pea3 IV sites, and a single point mutation is introduced in the Pea3 I site, rendering it a lower affinity Pea3 I site (thus engineering a vector in which all Pea3 sites are lower affinity). The sequence of the E1a enhancer region of the exemplary construct NV5 is set forth in SEQ ID NO:103.

In another embodiment, a vector is provided wherein, (in relation to the wild-type sequence), the flanking sequences around the Pea3 III and Pea3 II sites are altered to mimic the lower affinity Pea3 V and Pea3 IV sites, and the flanking regions of the Pea3 I site are altered, rendering it a lower affinity Pea3 I site (thus engineering a vector in which all Pea3 sites are lower affinity). The sequence of the E1a enhancer region of the exemplary construct NV6 is set forth in SEQ ID NO:104.

In another embodiment, a vector is provided wherein, (in relation to the wild-type sequence) the flanking sequences around the Pea3 I site are altered to produce a lower-affinity Pea3 I site, and the flanking sequences around the Pea3 II site is altered to mimic the lower affinity Pea3 IV site (thus engineering a vector in which all Pea3 sites except Pea3 III are lower affinity). The sequence of the E1a enhancer region of the exemplary construct NV7 is set forth in SEQ ID NO:105.

Example 11. In Vivo Demonstration of Reduction in Tumor Volume with Oncolytic Adenoviral Vectors Comprising E1a Enhancer Region Alterations and Encoding Immunomodulatory Polypeptides: Single Viruses with and without Antibody Treatment Materials and Methods

An anti-mPD-L1 antibody is, e.g., from BioXCell®, Catalog #BE0101 (Rat IgG2b). This antibody was used in the below experiments.

Virus samples are stored in 25 mM NaCl, 10 mM Tris Tris(hydroxymethyl)aminomethane), and 5% glycerol with a pH value of 8.0. Vials are stored protected from light at −80° C. On each day of dosing, one vial is thawed at room temperature for approximately 20 minutes. A single dose is 1×10⁹ pfu. Viruses to be combined with anti-PD-L1 in this Example include NV1-NV7 (having E1a enhancer region sequences set forth in SEQ ID Nos:99-105).

The mouse tumor model in this Example uses syngeneic immunocompetent mice. Female Jackson 12951 (12951/Sv1mJ) mice are used in this study. Mice are 6-7 weeks old on Day 1 of the experiment. The animals are fed irradiated Harlan 2918.15 Rodent Diet and water ad libitum. Animals are housed in static cages with Bed-O'Cobs™ bedding inside bioBubble® Clean Rooms that provide H.E.P.A filtered air into the bubble environment at 100 complete air changes per hour. All treatments, body weight determinations, and tumor measurements are carried out in the bubble environment. The environment is controlled to a temperature range of 70°±2° F. and a humidity range of 30-70%.

Cell Preparation

ADS-12 cells are grown in RPMI 1640 medium which is modified with 1% 100 mM Na pyruvate, 1% 200 mM L-glutamine, 1% 1M HEPES buffer, 1% of a 45% glucose solution and supplemented with 10% non-heat-inactivated Fetal Bovine Serum (FBS) and 1% 100× Penicillin/Streptomycin/L-Glutamine (PSG). The growth environment is maintained in an incubator with a 5% CO₂ atmosphere at 37° C. When expansion is complete, the cells (passage 7) are trypsinized using 0.25% trypsin/2.21 mM EDTA in HBSS solution. Following cell detachment, the trypsin is inactivated by dilution with complete growth medium and any clumps of cells are separated by pipetting. The cells are centrifuged at 200 rcf for 8 minutes at 4° C., the supernatant is aspirated, and the pellet is re-suspended in cold Dulbecco's Phosphate Buffered Saline (DPBS) by pipetting. An aliquot of the homogeneous cell suspension is diluted in a trypan blue solution and counted using a Luna automated cell counter. The cell suspension is centrifuged at 200 rcf for 8 minutes at 4° C. The supernatant is aspirated and the cell pellet is re-suspended in cold Dulbecco's Phosphate Buffered Saline (DPBS) to generate a final concentration of 1×10⁷trypan-excluding cells/ml. The cell suspension is maintained on wet ice during implantation. Following implantation, an aliquot of the remaining cells is diluted with a trypan blue solution and counted to determine the post-implantation cell viability.

Test animals are implanted subcutaneously on both flanks (on the back between the spine and the hip), the right flank on Day 0 and the left flank on Day 8, with 1×10⁶ cells in 0.1 ml of serum-free medium using a 28-gauge insulin syringe with a fixed needle.

All mice are sorted into study groups based on caliper measurement estimation of tumor burden on Day 15 when the mean tumor burden for all animals on the right flank is approximately 82 mm³ (range of group means, 75-90 mm³). The mice are distributed to ensure that the mean tumor burden on the right flank for all groups is within 10% of the overall mean tumor burden for the study population.

Results

The mean estimated right side tumor burden for all groups in the experiment on the first day of treatment is approximately 82 mm³ and all of the groups in the experiment are well-matched (range of group means, 75-90 mm³). All animals weigh at least 13.3 g at the initiation of therapy. Mean group body weights at first treatment are also well-matched (range, approximately 15.4-18.3 g). A tumor burden of 500 mm³ is chosen for evaluation of efficacy by tumor growth delay for the right and left tumors. The median Control Group tumor burdens will reach 500 mm³ on or about Day 47 for right tumors and on or about Day 43 for left tumors. The median tumor volume doubling times for the Control Group will be approximately 12 and 10 days for the right and left tumors, respectively.

Results of mouse inoculation and tumor growth will show that treatment of tumor bearing mice with oncolytic viruses comprising altered E1a regions and encoding various transgenes, with or without anti-PD-L1, show efficacy in reducing tumor volume.

Example 12. In Vivo Efficacy of Dual-Specificity Oncolytic Adenoviral Vectors in a Bilateral Tumor Model

Cancer immunotherapy is moving toward use of combinations to increase efficacy. Combining cancer immunotherapies can expand clinical benefits of existing approved monotherapies; however, systemically-administered combinations can produce excessive toxicity. Therefore, novel dual specificity oncolytic adenoviral vectors were developed having a transgene at both the E1 and the E3 regions in order to evaluate combinations of adenoviral-delivered immunomodulators to enhance systemic antitumor immunity. A summary of exemplary constructs contemplated for use by the methods disclosed herein (with additions to those disclosed in Table 5) is listed in Table 6.

TABLE 6 Exemplary E1/E3 Dual Transgene Constructs and Single Transgene Comparators (*) Name E1 E3 TRZ-001* CTLA-4 Del TRZ-009* CD70 intact TRZ-018* CD70 trimer Del TRZ-021* Empty IL-2 TRZ-032* IL-2 Empty (has 12.5K protein) TRZ-401 CTLA4 IL-12 TRZ-402 IL-10 Trap IL-12 TRZ-403 IL-7 IL-12 (has 12.5K protein) TRZ-404 CD70 IL-12 TRZ-408* Empty IL-12 TRZ-409 IL-12 IL-7 TRZ-411 OX40L IL-12 TRZ-412 IL-2 IL-12 TRZ-414 CD40L IL-12 TRZ-415 IL-12 CTLA4 TRZ-416 OX40L (trimer) IL-12 TRZ-417 CD40L (trimer) IL-12 TRZ-418 CD70 (trimer) IL-12 TRZ-421 IL-12 IL-2 TRZ-510 IL-7-P2A-IL-12 TRZ-202-like (no 12.5) TRZ-TBD IL-7-P2A-IL-12 IL-2

A bilateral tumor model was prepared using ADS-12 tumor cells as described in the Examples above (e.g., Example 7). 1×10⁶ ADS-12 tumor cells injected into primary (−2 days) and contralateral (0 days) flanks. At staging, mice are randomized based on contralateral tumors (88-150 mm³) and primary tumors (88-250 mm³). There are 8 mice/group, with tumors and body weight measured 3 times per week. An anti-PD-L1 (500 μg/dose) or an anti-PD-1 antibody (250 μg/dose) is administered i.p. to induce systemic T cell activation.

In this model, the virus is injected intratumorally (it) into primary tumors only; such injection leads to oncolysis, immune infiltration, and tumor shrinkage. The contralateral tumor is not injected, and no oncolysis occurs. Rather, tumor shrinkage is solely due to antigen-specific activated tumor infiltrating lymphocytes.

Results are shown in FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E, FIG. 9F, FIG. 9G, FIG. 9H, FIG. 9I, FIG. 9J, FIG. 9K, FIG. 9L, FIG. 9M, FIG. 9N, FIG. 9O, FIG. 9P, FIG. 9Q, FIG. 9R, FIG. 9S, FIG. 9T, FIG. 9U, FIG. 9V, FIG. 9W, FIG. 9X, FIG. 9Y, and FIG. 9Z. FIG. 9A is a cartoon of the contrast between the single transgene constructs (top) used in the virus mixing examples, and the dual transgene vectors used to make the E1/E3 transgene adenoviruses. FIG. 9B shows the results of mice injected i.t. with Empty virus+/−anti-PD-L1 or with TRZ-409 (IL-12/IL-7 dual transgene)+/−anti-PD-L1. The tumor volume of the injected tumor is shown in the left panel, and the tumor volume of the contralateral tumor is shown in the right panel. For each pair of arrows, the left arrow indicates virus injection and the right arrow indicates anti-PD-L1 injection. As can be seen for both the primary and contralateral tumors, TRZ-409 injection reduced tumor volume significantly compared to empty virus. The combination with anti-PD-L1 was slightly more efficacious in this study.

FIG. 9C shows the results of a second experiment using the inverse of TRZ-409, TRZ-403, in which the IL-7 transgene occupies the E1 region and the IL-12 gene occupies the E3 region (which has a stronger promoter than the E1). As can be seen in the Figure, TRZ-403 strongly inhibits primary and distant tumor growth, with or without anti-PD-L1. Plasma levels of IL-12 and IFN-γ (FIG. 9D) for all mice injected with TRZ-403 show that expression of the transgenes is well tolerated.

The study was extended for all mice having tumors over 500 mm³. Mice having primary tumors injected with TRZ-403 (IL-7+IL-12), TRZ-403+anti-PD-L1, TRZ-403+control IgG, or TRZ-409 (IL-12+IL-7), and controls including untreated mice and mice injected with empty vector TRZ-d19K with anti-PD-L1, anti-PD-1, or a control IgG are shown in FIG. 9E, which illustrates the survival as a percentage of mice still on study. As can be seen in the FIG. 9E, mice injected with TRZ-403 had a much higher percentage of survival over TRZ-409 at the time end of the study (70 days). Both TRZ-403 and TRZ-409 showed efficacy over the controls; all mice receiving control injections were deceased by day 56 of the study.

Next, a direct comparison was made between the dual transgene virus TRZ-403 (Il-7+IL-12) and a mixture of IL-7 and IL-12 single transgene viruses. As can be seen in FIG. 9F, in the primary tumor (left panel) TRZ-403 showed the most efficacy in reducing tumor volume, followed by the mixture of viruses. The single transgene IL-12 virus showed a small amount of efficacy by comparison. In the contralateral tumor, however (right panel) only the dual transgene virus, TRZ-403, reduced the tumor volume, showing significant superiority over the mixture of viruses.

Viruses containing two transgenes in the E1 region were then tested. FIG. 9G, FIG. 9H, FIG. 9I, FIG. 9J, FIG. 9K, FIG. 9L, FIG. 9M, FIG. 9N, FIG. 9O, FIG. 9P, FIG. 9Q, FIG. 9R, FIG. 9S, FIG. 9T, FIG. 9U, FIG. 9V, FIG. 9W, FIG. 9X, FIG. 9Y, and FIG. 9Z show comparisons of anti-tumor activity of various dual transgene viruses and controls, including comparison with antibody treatments. FIG. 9G, FIG. 9H, FIG. 9I, FIG. 9J, FIG. 9K, FIG. 9L, FIG. 9M, FIG. 9N, FIG. 9O, FIG. 9P, FIG. 9Q, FIG. 9R, FIG. 9S, FIG. 9T, FIG. 9U, FIG. 9V, FIG. 9W, FIG. 9X, FIG. 9Y, and FIG. 9Z show the primary tumor in the left panel and the contralateral tumor in the right panel. All viruses were administered intratumorally and the antibodies were administered intraperitoneally. Viral dose regimens vary and are tabulated below in Table 7. FIG. 9G shows the tumor activity in untreated animal controls; FIG. 9H shows TRZ000 (E1-Empty/E3 Intact); FIG. 9I, FIG. 9J, and FIG. 9K show TRZ002 (E1-mIL-12/E3 Del); FIG. 9L, FIG. 9M, and FIG. 9N show TRZ510 (E1-mIL-7-P2A-mIL-12/E3 Del); FIG. 9O, FIG. 9P, and FIG. 9Q show TRZ021 (E1-empty/E3-mIL-2); FIG. 9R, FIG. 9S, FIG. 9T, FIG. 9U, and FIG. 9V show TRZ421 (E1-mIL-12/E3-mIL-2); FIG. 9W shows the combination of TRZ002 (E1-mIL-12/E3 Del)+Anti-CTLA4 antibody (see Table 7 for doses); FIG. 9X shows anti-PD-1 antibody treatment alone, FIG. 9Y shows anti-CTLA4 antibody alone, and FIG. 9Z shows results of treatment with the combination of anti-CTLA-4 and anti-PD-1 antibodies (both from BioXCell).

Shown # Dose (PFU/inj + in Mice Construct Route Schedule μL/inj) FIG. 8 Untreated 9G 8 TRZ000 (E1-Empty/E3 Intact) IT Q4Dx3 1e9 9H 8 TRZ002 (E1-mIL-12/E3 Del) IT Q4Dx2 4e8 91 8 TRZ002 (E1-mIL-12/E3 Del) IT Q4Dx3 4e8 9J 8 TRZ002 (E1-mIL-12/E3 Del) IT Q4Dx3 1e9 9K 8 TRZ510 (E1-mIL-7-P2A-mIL-12/E3 IT Q4Dx2 4e8 9L Del) 8 TRZ510 (E1-mIL-7-P2A-mIL-12/E3 IT Q4Dx3 4e8 9M Del) 8 TRZ510 (E1-mIL-7-P2A-mIL-12/E3 IT Q4Dx3 1e9 9N Del) 8 TRZ021 (E1-empty/E3-mIL-2) IT Q4Dx2 4e8 90 8 TRZ021 (E1-empty/E3-mIL-2) IT Q4Dx3 4e8 9P 8 TRZ021 (E1-empty/E3-mIL-2) IT Q4Dx3 1e9 9Q 8 TRZ421 (E1-mIL-12/E3-mIL-2) IT Q4Dx1 4e8 9R 8 TRZ421 (E1-mIL-12/E3-mIL-2) IT Q4Dx2 4e8 9S 8 TRZ421 (E1-mIL-12/E3-mIL-2) IT Q4Dx3 4e8 9T 8 TRZ421 (E1-mIL-12/E3-mIL-2) IT Q4Dx2 1e9 9U 8 TRZ421 (E1-mIL-12/E3-mIL-2) IT Q4Dx3 1e9 9V 8 TRZ002 (E1-mIL-12/E3 Del) + Anti- IT + IP Q4Dx3 1e9 + 250 μL 9W CTLA4 Ab + Q4Dx3; 1d post virus 8 Anti-PD-1 Ab IP Q4Dx3 250 μL 9X 8 Anti-CTLA4 Ab IP Q4Dx3 250 μL 9Y 8 Anti-CTLA4 Ab + Anti-PD-1 Ab IP Q4Dx3 250 μL 9Z Dual constructs showed preliminary effectiveness at reducing tumor volume in mice, including TRZ421 and TRZ510.

Example 13. In Vitro Expression of Transgenes Cloned into E1 or E3 Site of Oncolytic Adenovirus

A549 cells (human lung adenocarcinoma, ATCC®, cat #CCL-85™) were grown in DMEM medium which is modified with 4.5 g/L D-glucose and 4 mM L-glutamine, and supplemented with 10% non-heat-inactivated fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S). The growth environment was maintained in an incubator with a 5% CO₂ atmosphere at 37° C. ADS-12 cells (murine lung adenocarcinoma cell line) were grown in RPMI 1640 medium which is modified with 1% 100 mM Na pyruvate, 1% 200 mM L-glutamine, 1% 1M HEPES buffer, 1% of a 45% glucose solution and supplemented with 10% non-heat-inactivated Fetal Bovine Serum (FBS) and 1% 100× Penicillin/Streptomycin/L-Glutamine (PSG). The growth environment was maintained in an incubator with a 5% CO₂ atmosphere at 37° C.

Sub-confluent A549 or ADS-12 cells were infected with TRZ000 (empty virus), TRZ510 (E1 IL-7-P2A-IL-12/E3), TRZ512 (E1 IL-12-P2A-IL-7/E3), TRZ403 (E1 IL-7/E3 IL-12), TRZ416 (E1 OX40L/E3 IL-12), or TRZ421 (E1 IL-12/E3 IL-2) at a multiplicity of infection (MOI) of 10 or 25. Forty-eight hours after infection, the supernatants were harvested, clarified by centrifugation, and assayed for protein expression by standard ELISA methods (mouse IL-12 (cat #M1270), mouse IL-2 (cat #M2000) and mouse IL-7 (cat #M7000) using Quantikine® kits, R&D Systems; mouse OX40L (cat #DY1236) using a DuoSet® kit, R&D Systems).

Results of measurement of secreted cytokines are shown in FIG. 10A FIG. 10B, FIG. 10C, FIG. 10D, FIG. 10E, FIG. 10F, and FIG. 10G. Expression of transgenes from the adenovirus E1 site is shown for IL-7 (FIG. 10A and FIG. 10B), OX40L (FIG. 10C), and IL-12 (FIG. 10D and FIG. 10E). Expression of transgenes from the E3 site is shown for IL-12 (FIG. 10F) and IL-2 (FIG. 10G). As can be seen in the Figures, all transgenes were expressed well from either the E1 or E3 site.

Example 14. In Vitro Functional Analysis of Transgenes Cloned into E1 or E3 Site of Oncolytic Adenovirus

A549 cells were grown in DMEM medium which is modified with 4.5 g/L D-glucose and 4 mM L-glutamine, and supplemented with 10% non-heat-inactivated fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S). The growth environment was maintained in an incubator with a 5% CO₂ atmosphere at 37° C. In parallel, HEK-Blue™ IL-12 cells (modified human embryonic kidney (HEK) 293 cells; InvivoGen, cat #HKB-IL12) were grown in DMEM medium which is modified with 4.5 g/L D-glucose and 4 mM L-glutamine, and supplemented with 10% fetal bovine serum (FBS), 50 ng/ml of recombinant human IL-12 and 1% Penicillin/Streptomycin (P/S). The growth environment was maintained in an incubator with a 5% CO₂ atmosphere at 37° C. CTLL-2 cells (mouse lymphocyte cells, ATCC®, cat #TIB-214™) were grown in RPMI medium which is modified with 2 mM glutamine, and supplemented with 10% fetal bovine serum (FBS), and 20 IU/ml human recombinant IL-2. The growth environment was maintained in an incubator with a 5% CO₂ atmosphere at 37° C.

Sub-confluent A549 cells were infected with TRZ510 (E1 IL-7-P2A-IL-12/E3), TRZ512 (E1 IL-12-P2A-IL-7/E3), TRZ403 (E1 IL-7/E3 IL-12), and TRZ416 (E1 OX40L/E3 IL-12) at a multiplicity of infection (MOI) of 10 or 25. Forty-eight hours after infection, the supernatants were harvested, clarified by centrifugation, and assayed for biological function of secreted IL-12 using HEK-Blue™ IL-12 cells. The HEK-Blue IL-12 cells were washed twice to remove excess recombinant human IL-12 and plated in each test well at 2×10⁵ cells/well in 180 μL using 96-well round bottom plates. Serial dilutions of A549 supernatants from TRZ416-infected A549 cells were prepared at a 1:3 dilution and 20 μL were added to the 180 μL with HEK-Blue IL-12 cells. The last well with HEK-Blue IL-12 cells received 20 μL of medium only as negative control. Twenty-four hours later 20 μL of HEK-Blue IL-12 cell supernatant was added to 180 μL of QUANTI-Blue™ (InvivoGen, cat #HKB-IL12) in a 96 well ELISA plate and incubated for 30 min at 37° C. Secreted embryonic alkaline phosphatase (SEAP) levels were measured using a spectrophotometer at 620 nm. Alternatively, above described clarified supernatants from A549 cells were assayed for biological function of secreted IL-2 using CTLL-2 cells. CTLL-2 cells were washed twice to remove excess recombinant human IL-2 and plated in each test well at 5×10⁴ cells/well in 100 μL using 96-well round bottom plates. Serial dilutions of A549 supernatants from TRZ421-infected A549 cells were prepared at a 1:3 dilution and 100 μL were added to the 100 μL with CTLL-2 cells. The last well with CTLL-2 cells received 100 μL of medium only as negative control. Seventy-two hours later 100 μL of CellTiter-Glo® (Promega, cat #G9241) was added to the induced CTLL-2 cells in 100 μL and incubated for 10 min at room temperature. Combined CellTiter-Glo and CTLL-2 cells were transferred to a 96-well clear bottom plate for luminescence reading. Luminescence was recorded at an integration time of 0.5 sec/well.

Results of functional analysis of secreted cytokines are shown in FIG. 11A FIG. 11B, FIG. 11C, FIG. 11D, and FIG. 11E. Function of IL-12 transgenes in HEK-Blue IL-12 cells is shown for IL-12 in supernatants expressed from the E1 region (FIG. 11A, TRZ510; FIG. 11B, TRZ512) and from the E3 region (FIG. 11C; TRZ403; FIG. 11D; TRZ416). Function of IL-2 transgene in CTLL-2 cells is shown for supernatants expressed from the E3 region is shown in FIG. 11E (TRZ421). As can be seen in FIG. 11A FIG. 11B, FIG. 11C, FIG. 11D, and FIG. 11E, all transgenes were functional when expressed from the E1 or E3 sites, including those connected with a P2A sequence.

ENDNOTES

Although the invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

All publications, databases, GenBank sequences, patents, and patent applications cited in this specification are herein incorporated by reference as if each specifically and individually indicated to be incorporated by reference.

APPENDIX A SEQUENCE REFERENCE Accession SEQ Number(s) ID or NO: Molecule description Sequence 1 hGITR-Ligand NP_005083 MTLHPSPITCEFLFSTALISPKMCLSHLENMPLSHSRTQGAQRS (GITR-L; SWKLWLFCSIVMLLFLCSFSWLIFIFLQLETAKEPCMAKFGPL TNFSF18) PSKWQMASSEPPCVNKVSDWKLEILQNGLYLIY GQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGT YELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFIS 2 mGITR-Ligand NM_183391 MEEMPLRESSPQRAERCKKSWLLCIVALLLMLLCSLGTLIYTS (GITR-L; LKPTAIESCMVKFELSSSKWHMTSPKPHCVNTTSDGKLKILQS TNFSF18) GTYLIYGQVIPVDKKYIKDNAPFVVQIYKKNDVLQTLMNDFQ ILPIGGVYELHAGDNIYLKFNSKDHIQKTNTYWGIILMPDLPFI S 3 hCD28 ligand EAW79565 MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEV or agonist KEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMN (CD80) IWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLK YEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTS GGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFN MTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFP DNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRES VRPV 4 hTNFα CAA78745 MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAG ATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKP VAHVVANPQAEGQLQWLNRRANALLANGVELR DNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSY QTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKG DRLSAEINRPDYLDFAESGQVYFGIIAL 5 Non-cleavable NM_013693 MIETYSQPSPRSVATGLPASMKIFMYLLTVFLITQMIGSVLFAV mTNFα YLHRRLDKVEEEVNLHEDFVFIKKLKRCNKGEGSLSLLNCEE MRRQFEDLVKDITLNKEEKKEDEDPVAHVVANHQVEEQLEW LSQRANALLANGMDLKDNQLVVPADGLYLVYSQVLFKGQG CPDYVLLTHTVSRFAISYQEKVNLLSAVKSPCPKDTPEGAELK PWYEPIYLGGVFQLEKGDQLSAEVNLPKYLDFAESGQVYFGII AL 6 mGM-CSF NM_009969 MWLQNLLFLGIVVYSLSAPTRSPITVTRPWKHVEAIKEALNLL DDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFT KLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTF LTDIPFECKKPGQK 7 hICOS ligand NP_056074 MRLGSPGLLFLLFSSLRADTQEKEVRAMVGSDVELSCACPEG or agonist SRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRA LMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSL GFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELTFTCTSING YPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLR IARTPSVNIGCCIENVLLQQNLTVGSQTGNDIGERD KITENPVSTGEKNAATWSILAVLCLLVVVAVAIGWVCRDRCL QHSYAGAWAVSPETELTGHV 8 h4-1BB ligand AAA53134 MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLL or agonist AAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAG LLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSL TGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSV SLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRL LHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRV TPEIPAGLPSPRSE 9 hOX40 ligand CAE11757 MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDR or agonist CCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKP CTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYK PGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSS DAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTR PVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLL RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI 10 mOX40 ligand NM_009452 MEGEGVQPLDENLENGSRPRFKWKKTLRLVVSGIKGAGMLL CFIYVCLQLSSSPAKDPPIQRLRGAVTRCEDGQLFISSYKNEYQ TMEVQNNSVVIKCDGLYITYLKGSFFQEVKIDLHFREDHNPISI PMLNDGRRIVFTVVASLAFKDKVYLTVNAPDTLCEHLQINDG ELIVVQLTPGYCAPEGSYHSTVNQVPL 11 hCD40 ligand NP_000065 MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAV or agonist YLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEI KSQFEGFVKDIMLNKEETKKENSFEMQKGDQNP QIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLT VKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILL RAANTHSSAKPCGQQSIHLGGVFELQPGASVFVN VTDPSQVSHGTGFTSFGLLKL 12 mCD40 ligand NM_011616 MIETYSQPSPRSVATGLPASMKIFMYLLTVFLITQMIGSVLFAV YLHRRLDKVEEEVNLHEDFVFIKKLKRCNKGEGSLSLLNCEE MRRQFEDLVKDITLNKEEKKENSFEMQRGDEDPQIAAHVVSE ANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLY YVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTH SSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFS SFGLLKL 13 hCD27 ligand AAA36175 MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFA or agonist QAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPALG RSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTA SRHEIPTTLAVGICSPASRSISLLRLSFHQGCTIVSQRLTPLARG DTLCTNLTGTLLPSRNTDETFFGVQWVRP 14 mCD70 ligand NM_011617 MPEEGRPCPWVRWSGTAFQRQWPWLLLVVFITVFCCWFHCS or agonist GLLSKQQQRLLEHPEPHTAELQLNLTVPRKDPTLRWGAGPAL GRSFTHGPELEEGHLRIHQDGLYRLHIQVTLANCSSPGSTLQH RATLAVGICSPAAHGISLLRGRFGQDCTVALQRLTYLVHGDV LCTNLTLPLLPSRNADETFFGVQWICP 15 hInterleukin-2 AAB46883 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQ (IL-2) MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK PLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE TTFMCEYADETATIVEFLNRWITFCQSIISTLT 16 hInterleukin-7 AAH47698 MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMV (IL-7) SIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAAR KLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQ VKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQE IKTCWNKILMGTKEH 17 murine MM_008371.5 MFHVSFRYIFGIPPLILVLLPVTSSECHIKDKEGKAYESVLMISI Interleukin-7 DELDKMTGTDSNCPNNEPNFFRKHVCDDTKEAAFLNRAARK (IL-7) LKQFLKMNISEEFNVHLLTVSQGTQTLVNCTSKEEKNVKEQK KNDACFLKRLLREIKTCWNKILKGSI 18 Interleukin-12 AAD16432 MWPPGSASQPPPSPAAATGLHPAARPVSLQCRLSMCPARSLL (IL-12) alpha LVATLVLLDHLSLARNLPVATPDPGMFPCLHEISQNLLRAVSN subunit MLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACL PLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYED LKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQAL NFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMS YLNAS 19 hInterleukin-12 NP_005526 MEPLVTWVVPLLFLFLLSRQGAACRTSECCFQDPPYPDADSG (IL-12) SASGPRDLRCYRISSDRYECSWQYEGPTAGVSHFLRCCLSSGR receptor CCYFAAGSATRLQFSDQAGVSVLYTVTLWVESWAR subunit beta-1 NQTEKSPEVTLQLYNSVKYEPPLGDIKVSKLAGQLRMEWETP isoform DNQVGAEVQFRHRTPSSPWKLGDCGPQDDDTESCLCPLEMN precursor VAQEFQLRRRQLGSQGSSWSKWSSPVCVPPENPPQPQ VRFSVEQLGQDGRRRLTLKEQPTQLELPEGCQGLAPGTEVTY RLQLHMLSCPCKAKATRTLHLGKMPYLSGAAYNVAVISSNQ FGPGLNQTWHIPADTHTEPVALNISVGTNGTTMYWPA RAQSMTYCIEWQPVGQDGGLATCSLTAPQDPDPAGMATYSW SRESGAMGQEKCYYITIFASAHPEKLTLWSTVLSTYHFGGNA SAAGTPFIEWSVKNHSLDSVSVDWAPSLLSTCPGVLKE YVVRCRDEDSKQVSEHPVQPTETQVTLSGLRAGVAYTVQVR ADTAWLRGVWSQPQRFSIEVQVSDWLIFFASLGSFLSILLVGV LGYLGLNRAARHLCPPLPTPCASSAIEFPGGKETWQ WINPVDFQEEASLQEALVVEMSWDKGERTEPLEKTELPEGAP ELALDTELSLEDGDRCKAKM 20 murine N/A MCPQKLTISWFAIVLLVSPLMAMWELEKDVYVVEVDWTPDA Interleukin-12 PGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLD fusion AGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFL polypeptide KCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCG MASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALE ARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWE YPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLV EKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSG GGGSGGGGSGGGGSRVIPVSGPARCLSQSRNLLKTTDDMVKT AREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCL ATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQ AINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKP PVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA 21 hIL-15 CAA62616 MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPK TEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTA MKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTES GCKECEELEEKNIKEFLQSFVHIVQMFINTS 22 mIL-15 hybrid N/A MKILKPYMRNTSISCYLCFLLNSHFLTEAGIHVFILGCVSVGLP KTEANWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAM NCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAE SGCKECEELEEKTFTEFLQSFIRIVQMFINTSGSGATNFSLLKQ AGDVEENPGPGTTCPPPVSIEHADIRVKNYSVNSRERYVCNSG FKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPV PTVVTPKVTSQPESPSPSAKEPEAFSPKSDTAMTTETAIMPGSR LTPSQTTSAGTTGTGSHKSSRAPSLAATMTLEPTASTSLRITEIS PHSSKMTK 23 hIL-10 receptor NP_001549 MLPCLVVLLAALLSLRLGSDAHGTELPSPPSVWFEAEFFHHIL subunit alpha HWTPIPNQSESTCYEVALLRYGIESWNSISNCSQTLSYDLTAV precursor TLDLYHSNGYRARVRAVDGSRHSNWTVTNTRFSV DEVTLTVGSVNLEIHNGFILGKIQLPRPKMAPANDTYESIFSHF REYEIAIRKVPGNFTFTHKKVKHENFSLLTSGEVGEFCVQVKP SVASRSNKGMWSKEECISLTRQYFTVTNVIIFF AFVLLLSGALAYCLALQLYVRRRKKLPSVLLFKKPSPFIFISQR PSPETQDTIHPLDEEAFLKVSPELKNLDLHGSTDSGFGSTKPSL QTEEPQFLLPDPHPQADRTLGNREPPVLGDSC SSGSSNSTDSGICLQEPSLSPSTGPTWEQQVGSNSRGQDDSGID LVQNSEGRAGDTQGGSALGHHSPPEPEVPGEEDPAAVAFQGY LRQTRCAEEKATKTGCLEEESPLTDGLGPKFGRC LVDEAGLHPPALAKGYLKQDPLEMTLASSGAPTGQWNQPTE EWSLLALSSCSDLGISDWSFAHDLAPLGCVAAPGGLLGSFNS DLVTLPLISSLQSSE 24 mIL-10R Trap N/A MLSRLLPFLVTISSLSLEFIAYGTELPSPSYVWFEARFFQHILH WKPIPNQSESTYYEVALKQYGNSTWNDIHICRKAQALSCDLT TFTLDLYHRSYGYRARVRAVDNSQYSNWTTTETRFTVDEVIL TVDSVTLKAMDGIIYGTIHPPRPTITPAGDEYEQVFKDLRVYKI SIRKFSELKNATKRVKQETFTLTVPIGVRKFCVKVLPRLESRIN KAEWSEEQCLLITTEQYFTVTNLSHPASSTKVDKKIVPRDCGC KPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPE VQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWL NGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQ MAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIM DTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEK SLSHSPGK 25 hIL-27 NP_004834 MRGGRGAPFWLWPLPKLALLPLLWVLFQRTRPQGSAGPLQC Receptor YGVGPLGDLNCSWEPLGDLGAPSELHLQSQKYRSNKTQTVA subunit alpha VAAGRSWVAIPREQLTMSDKLLVWGTKAGQPLWPPVFV precursor NLETQMKPNAPRLGPDVDFSEDDPLEATVHWAPPTWPSHKV LICQFHYRRCQEAAWTLLEPELKTIPLTPVEIQDLELATGYKV YGRCRMEKEEDLWGEWSPILSFQTPPSAPKDVWVSG NLCGTPGGEEPLLLWKAPGPCVQVSYKVWFWVGGRELSPEG ITCCCSLIPSGAEWARVSAVNATSWEPLTNLSLVCLDSASAPR SVAVSSIAGSTELLVTWQPGPGEPLEHVVDWARDGD PLEKLNWVRLPPGNLSALLPGNFTVGVPYRITVTAVSASGLAS ASSVWGFREELAPLVGPTLWRLQDAPPGTPAIAWGEVPRHQL RGHLTHYTLCAQSGTSPSVCMNVSGNTQSVTLPDL PWGPCELWVTASTIAGQGPPGPILRLHLPDNTLRWKVLPGILF LWGLFLLGCGLSLATSGRCYHLRHKVLPRWVWEKVPDPANS SSGQPHMEQVPEAQPLGDLPILEVEEMEPPPVMESS QPAQATAPLDSGYEKHFLPTPEELGLLGPPRPQVLA 26 hIL-13 AAH96140 MVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHK VSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN 27 hIL-17 AAC50341 MTPGKTSLVSLLLLLSLEAIVKAGITIPRNPGCPNSEDKNFPRT VMVNLNIHNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERY PSVIWEAKCRHLGCINADGNVDYHMNSVPIQQEIL VLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA 28 hIL-33 NP_ MKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQKAKEVCP Isoform a 001300974 MYFMKLRSGLMIKKEACYFRRETTKRPSLKTGRKHKRHLVL AACQQQSTVECFAFGISGVQKYTRALHDSSITGISPIT EYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSY YESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVE LHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFI GVKDNHLALIKVDSSENLCTENILFKLSET 29 hIL-33 NP_ MKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQKAKEVCP Isoform a 001300973 MYFMKLRSGLMIKKEACYFRRETTKRPSLKTGRKHKRHLVL AACQQQSTVECFAFGISGVQKYTRALHDSSITGISPIT EYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSY YESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVE LHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFI GVKDNHLALIKVDSSENLCTENILFKLSET 30 hIL-33 NP_ MKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQKAKEVCP Isoform b 001186569 MYFMKLRSGLMIKKEACYFRRETTKRPSLKTGRKHKRHLVL AACQQQSTVECFAFGISGVQKYTRALHDSSITDKVLLS YYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHS VELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKD NHLALIKVDSSENLCTENILFKLSET 31 hIL-33 NP_ MKPKMKYSTNKISTAKWKNTASKALCFKLGNKVLLSYYESQ Isoform c 001186570 HPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHK CEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNH LALIKVDSSENLCTENILFKLSET 32 hIL-33 NP_ MKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQKAKEVCP Isoform d 001300975; MYFMKLRSGLMIKKEACYFRRETTKRPSLKTGRKHKRHLVL NP_ AACQQQSTVECFAFGISGVQKYTRALHDSSITEYLASL 001300976 STYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHP SNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCE KPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNH LALIKVDSSENLCTENILFKLSET 33 hIL-33 NP_ MKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQKAKEVCP Isoform e 001300977 MYFMKLRSGLMIKKEACYFRRETTKRPSLKTGISPITEYLASL STYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLS YYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHS VELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKD NHLALIKVDSSENLCTENILFKLSET 34 hIFN-γ AAB59534 MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNAGH SDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFK DDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTN YSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGR RASQ 35 Flagellin p06179 MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKD DAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEIN NNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQ TQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDT LNVQQKYKVSDTAATVTGYADTTIALDNSTFKASATGLGGT DQKIDGDLKFDDTTGKYYAKVTVTGGTGKDGYYEVSVDKT NGEVTLAGGATSPLTGGLPATATEDVKNVQVANADLTEAKA ALTAAGVTGTASVVKMSYTDNNGKTIDGGLAVKVGDDYYS ATQNKDGSISINTTKYTADDGTSKTALNKLGGADGKTEVVSI GGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQKIDAALA QVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARSRIEDSDY ATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR 36 anti-CTLA4 N/A MDMRVPAQLLGLLLLWLRGARCDIVMTQTTLSLPVSLGDQA (CTLA-4 SISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSG antagonist VPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGG antibody) GTKLEIKRADAAPTVSGSGGGSGGGSGGGSEAKLQESGPVLV KPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGVINP YNGDTSYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVY YCARYYGSWFAYWGQGTLITVSTEPRGPTIKPCPPCKCPAPN LLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISW FVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKE FKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKK QVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDG SYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHEITTKSFSR TPGK 37 murine IL-2 DNA TATCACCCTTGCTAATCACTCCTCACAGTGACCTCAAGTCC (NM_008366.3) TGCAGGCATGTACAGCATGCAGCTCGCATCCTGTGTCACAT TGACACTTGTGCTCCTTGTCAACAGCGCACCCACTTCAAGC TCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAGC AGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTT GATGGACCTACAGGAGCTCCTGAGCAGGATGGAGAATTAC AGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTA CTTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGC CTAGAAGATGAACTTGGACCTCTGCGGCATGTTCTGGATTT GACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAAT TTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGG GCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCA GCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTG TCAAAGCATCATCTCAACAAGCCCTCAATAACTATGTACCT CCTGCTTACAACACATAAGGCTCTCTATTTATTTAAATATT TAACTTTAATTTATTTTTGGATGTATTGTTTACTATCTTTTG TAACTACTAGTCTTCAGATGATAAATATGGATCTTTAAAGA TTCTTTTTGTAAGCCCCAAGGGCTCAAAAATGTTTTAAACT ATTTATCTGAAATTATTTATTATATTGAATTGTTAAATATCA TGTGTAGGTAGACTCATTAATAAAAGTATTTAGATGATTCA AATATAAATAAGCTCAGATGTCTGTCATTTTTAGGACAGCA CAAAGTAAGCGCTAAAATAACTTCTCAGTTATTCCTGTGAA CTCTATGTTAATCAGTGTTTTCAAGAAATAAAGCTCTCCTC TAAAAAAAAAAAAAAA 38 murine IL-2 amino acid MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQ CAA25909 QQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQ ATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRV TVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ 39 murine Xcl1 DNA AGCCCAGCAAGACCTCAGCCATGAGACTTCTCCTCCTGACT (GenBank: TTCCTGGGAGTCTGCTGCCTCACCCCATGGGTTGTGGAAGG BC062249.1) TGTGGGGACTGAAGTCCTAGAAGAGAGTAGCTGTGTGAAC TTACAAACCCAGCGGCTGCCAGTTCAAAAAATCAAGACCT ATATCATCTGGGAGGGGGCCATGAGAGCTGTAATTTTTGTC ACCAAACGAGGACTAAAAATTTGTGCTGATCCAGAAGCCA AATGGGTGAAAGCAGCGATCAAGACTGTGGATGGCAGGGC CAGTACCAGAAAGAACATGGCTGAAACTGTTCCCACAGGA GCCCAGAGGTCCACCAGCACAGCGATAACCCTGACTGGGT AACAGCCTCCAGGACAATGTTTCCTCACTCGTTAAGCAGCT CATCTCAGTTCCCAAACCCATTGCACAAATACTTATTTTTA TTTTTAACGACATTCACATTCATTTCAAATGTTATAAGTAA TAAATATTTATTATTGATGAAAAAAAAAAAAAAAAAAAA 40 murine Xcl1 amino acid MRLLLLTFLGVCCLTPWVVEGVGTEVLEESSCVNLQTQRLPV (GenBank: QKIKTYIIWEGAMRAVIFVTKRGLKICADPEAKWVKAAIKTV BC062249.1) DGRASTRKNMAETVPTGAQRSTSTAITLTG 41 Murine 4- (DNA) GAGACGTGCACTGACCGACCGTGGTAATGGACCAGCACAC 1BBL GenBank: ACTTGATGTGGAGGATACCGCGGATGCCAGACATCCAGCA (TNFSF9) BC138767.1 GGTACTTCGTGCCCCTCGGATGCGGCGCTCCTCAGAGATA CCGGGCTCCTCGCGGACGCTGCGCTCCTCTCAGATACTGTG CGCCCCACAAATGCCGCGCTCCCCACGGATGCTGCCTACCC TGCGGTTAATGTTCGGGATCGCGAGGCCGCGTGGCCGCCT GCACTGAACTTCTGTTCCCGCCACCCAAAGCTCTATGGCCT AGTCGCTTTGGTTTTGCTGCTTCTGATCGCCGCCTGTGTTCC TATCTTCACCCGCACCGAGCCTCGGCCAGCGCTCACAATCA CCACCTCGCCCAACCTGGGTACCCGAGAGAATAATGCAGA CCAGGTCACCCCTGTTTCCCACATTGGCTGCCCCAACACTA CACAACAGGGCTCTCCTGTGTTCGCCAAGCTACTGGCTAAA AACCAAGCATCGTTGTGCAATACAACTCTGAACTGGCACA GCCAAGATGGAGCTGGGAGCTCATACCTATCTCAAGGTCT GAGGTACGAAGAAGACAAAAAGGAGTTGGTGGTAGACAG TCCCGGGCTCTACTACGTATTTTTGGAACTGAAGCTCAGTC CAACATTCACAAACACAGGCCACAAGGTGCAGGGCTGGGT CTCTCTTGTTTTGCAAGCAAAGCCTCAGGTAGATGACTTTG ACAACTTGGCCCTGACAGTGGAACTGTTCCCTTGCTCCATG GAGAACAAGTTAGTGGACCGTTCCTGGAGTCAACTGTTGC TCCTGAAGGCTGGCCACCGCCTCAGTGTGGGTCTGAGGGC TTATCTGCATGGAGCCCAGGATGCATACAGAGACTGGGAG CTGTCTTATCCCAACACCACCAGCTTTGGACTCTTTCTTGTG AAACCCGACAACCCATGGGAATGAGAACTATCCTTCTTGT GACTCCTAGTTGCTAAGTCCTCAAGCTGCTATGTTTTATGG GGTCTGAGCAGGGGT 42 Murine 4- (protein) MDQHTLDVEDTADARHPAGTSCPSDAALLRDTGLLADAALL 1BBL GenBank: SDTVRPTNAALPTDAAYPAVNVRDREAAWPPALNFCSRHPK (TNFSF9) BC138767.1 LYGLVALVLLLLIAACVPIFTRTEPRPALTITTSPNLGTRENNA DQVTPVSHIGCPNTTQQGSPVFAKLLAKNQASLCNTTLNWHS QDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKLSPTF TNTGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPCSMENK LVDRSWSQLLLLKAGHRLSVGLRAYLHGAQDAYRDWELSYP NTTSFGLFLVKPDNPWE 43 Mouse 4-1BBL with human MDMRVPAQLLGLLLLWLRGARCRMKQIEDKIEEILSKIYHIEN trimeric version IGKV1-39 EIARIKKLIGERGGGSGGGSGGGSRTEPRPALTITTSPNLGTRE kappa LC NNADQVTPVSHIGCPNTTQQGSPVFAKLLAKNQASLCNTTLN signal WHSQDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKL sequence, SPTFTNTGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPCS trimerization MENKLVDRSWSQLLLLKAGHRLSVGLRAYLHGAQDAYRDW domain from ELSYPNTTSFGLFLVKPDNPWE** yeast, and Linker 1, followed by 4-1BBL (TNFSF9, Acc# P41274) residues 104- 309 (minus ECD and transmembrane domain) 44 murine 4- residues 104- RTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGSP 1BBL 209 (minus VFAKLLAKNQASLCNTTLNWHSQDGAGSSYLSQGLRYEEDK (TNFSF9, ECD and KELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVLQAKP p41274) trans- QVDDFDNLALTVELFPCSMENKLVDRSWSQLLLLKAGHRLS membrane VGLRAYLHGAQDAYRDWELSYPNTTSFGLFLVKPDNPWE** domain) 45 Linker 1 15-mer GGGGSGGGGSGGGGS 46 TRZ202 (hIL- AA Sequence MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDA 12 Insert) PGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFG DAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKN KTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGV TCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEV MVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVE VSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTS ATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGG SGGGGSRNLPVATPDPGMFPCLIAHSQNLLRAVSNMLQKARQ TLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRE TSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNA KLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEE PDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS** 47 hIL-12 p40β AF180563 MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDA PGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFG DAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKN KTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGV TCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEV MVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVE VSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTS ATVICRKNASISVRAQDRYYSSSWSEWASVPCS 48 hIL-12 p35α AA Sequence RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPC of Accession TSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGS #AF 101062.1 CLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPK (native start RQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKI codon and KLCILLHAFRIRAVTIDRVMSYLNAS** signal sequence removed) 49 (TRZ015) AA GTCGACGCCACCMDMRVPAQLLGLLLLWLRGARCRMKQI hGITRL insert, Sequence, EDKIEEILSKIYHIENEIARIKKLIGERGGGSGGGSGGGSETAKE Trimeric including PCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIY version Kozak GQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGT sequences. YELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFIS** RE sites, and CTCGAG stop codons 50 hIGKV1-39 AA Sequence MDMRVPAQLLGLLLLWLRGARC kappa LC signal sequence 51 Trimerization AA Sequence RMKQIEDKIEEILSKIYHIENEIARIKKLIGER domain (yeast) 52 GITRL minus AA Sequence ETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQN ECD and GLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQ transmembrane NVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFIS** domains 53 Linker 2 12-mer GGGSGGGSGGGS 54 TRZ006:hIL- hIL-15Rα- MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHAD 15 hybrid Linker-hIL- IWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAH 15 WTTPSLKCIRDPALVHQRPAPPSTVTTASGGSGGGGSGGGSG GGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVT AMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTE SGCKECEELEEKNIKEFLQSFVHIVQMFINTS** 55 hIL-15Rα AA Sequence MAPRRARGCRTLGLPALLLLLLLRPPATRG endogenous signal peptide 56 Sushi domain AA Sequence MSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLN from IL-15Rα KATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTA 57 mature hIL-15 AA Sequence NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKC FLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCK ECEELEEKNIKEFLQSFVHIVQMFINTS 58 TRZ201: Orientation 2 MDMRVPAQLLGLLLLWLRGARCEIVLTQSPATLSLSPGERAT haCTLA4 ANT2054 LSCSASSSISYMHWFQQRPGQSPRRWIYDTSKLASGVPARFSG scFv-IgG1 Fc SGSGTDYTLTISSLEPEDFATYYCHQRTSYPLTFGQGTKLEIKG GGGSGGGGSGGGGSQVQLVQSGAELKKPGASVKVSCKASGY TFTSYWINWIRQAPGQGLEWIGRIAPGSGTTYYNEVFKGRVTI TVDKSTSTAYMELSSLRSEDTAVYFCARGDYGSYWGQGTLV TVSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK** 59 Kappa variable AA Sequence EIVLTQSPATLSLSPGERATLSCSASSSISYMHWFQQRPGQSPR RWIYDTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFATYYC HQRTSYPLTFGQGTKLEIK 60 Heavy variable AA Sequence QVQLVQSGAELKKPGASVKVSCKASGYTFTSYWINWIRQAP GQGLEWIGRIAPGSGTTYYNEVFKGRVTITVDKSTSTAYMEL SSLRSEDTAVYFCARGDYGSYWGQGTLVTVSS 61 human Fc IgG1 AA Sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK** 62 TRZ106 hIL-15 MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHAD hybrid (hIL- IWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAH 15Rα-Linker WTTPSLKCIRDPALVHQRPAPPSTVTTASGGSGGGGSGGGSG 2-hIL-15) GGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVT (hIL-15Rα AMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTE endogenous SGCKECEELEEKNIKEFLQSFVHIVQMFINTS** signal peptide, Sushi domain from IL- 15Rα, linker, mature hIL- 15) 63 hIL-15Rα MAPRRARGCRTLGLPALLLLLLLRPPATRG endogenous signal peptide 64 mature hIL-15 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKC FLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCK ECEELEEKNIKEFLQSFVHIVQMFINTS 65 TRZ108: full length MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAV hCD40 monomeric YLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEI version, KSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISE membrane ASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLY bound YIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHS SAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSF GLLKL** 66 TRZ111: full length MERVQPLEENVGNAARPRFERNKLLLVASVIQGLGLLLCFTYI hOX40L monomeric CLHFSALQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMK variant 1 version, VQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLK membrane KVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELI bound LIHQNPGEFCVL** 67 TRZ114: hIL- Extracellular MLPCLVVLLAALLSLRLGSDAHGTELPSPPSVWFEAEFFHHIL 10RTrap domain of HWTPIPNQSESTCYEVALLRYGIESWNSISNCSQTLSYDLTAV Version2 hIL-10Rα TLDLYHSNGYRARVRAVDGSRHSNWTVTNTRFSVDEVTLTV with GSVNLEIHNGFILGKIQLPRPKMAPANDTYESIFSHFREYEIAIR endogenous KVPGNFTFTHKKVKHENFSLLTSGEVGEFCVQVKPSVASRSN signal KGMWSKEECISLTRQYFTVTNDKTHTCPPCPAPELLGGPSVFL sequence, FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV without stop HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS and human NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC Fc IgG1 LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK** 68 Extracellular HGTELPSPPSVWFEAEFFHHILHWTPIPNQSESTCYEVALLRY domain of hIL- GIESWNSISNCSQTLSYDLTAVTLDLYHSNGYRARVRAVDGS 10Rα RHSNWTVTNTRFSVDEVTLTVGSVNLEIHNGFILGKIQLPRPK MAPANDTYESIFSHFREYEIAIRKVPGNFTFTHKKVKHENFSL LTSGEVGEFCVQVKPSVASRSNKGMWSKEECISLTRQYFTVT N 69 endogenous MLPCLVVLLAALLSLRLGSDA signal sequence in TRZ114 70 human Fc IgG1 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK** 71 TRZ116: Trimeric MDMRVPAQLLGLLLLWLRGARCRMKQIEDKIEEILSKIYHIEN hOX40L version EIARIKKLIGERGGGSGGGSGGGSETAKEPCMAKFGPLPSKW w/human QMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDV IGKV 1-39 APFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFN kappa LC SEHQVLKNNTYWGIILLANPQFIS** signal sequence, trimerization domain from yeast, and Linker 1, followed by OX40L minus ECD and trans- membrane domains 72 hOX40L minus QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSV ECD and IINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNS transmembrane LMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGE domains FCVL** 73 TRZ117: Trimeric MDMRVPAQLLGLLLLWLRGARCRMKQIEDKIEEILSKIYHIEN hCD40L version EIARIKKLIGERGGGSGGGSGGGSMQKGDQNPQIAAHVISEAS w/human SKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIY IGKV 1-39 AQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAK kappa LC PCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLL signal KL** sequence, trimerization domain from yeast, and Linker 1, followed by CD40L minus ECD and trans- membrane domains 76 hCD40L minus MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLV ECD and TLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLK trans- SPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFV membrane domains NVTDPSQVSHGTGFTSFGLLKL** 77 TRZ307: hIL-7 MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMV variant 1 SIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAAR KLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAA LGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKIL MGTKEH** hIL-7 MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMV SIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAAR KLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAA LGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKIL MGTKEH** 78 TR2304: MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLL hGM-CSF NLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRG SLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLK DFLLVIPFDCWEPVQE** 79 hGM-C SF MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLL NLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRG SLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLK DFLLVIPFDCWEPVQE** 80 TRZ309: (residue in MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFA hCD70 bold to be QAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPALG removed) RSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHP TTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCT NLTGTLLPSRNTDETFFGVQWVRP** 81 hCD70 (residue in MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFA bold to be QAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPALG modified to RSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHP avoid TTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCT introducing NLTGTLLPSRNTDETFFGVQWVRP** new Xho I site) 82 E3 14.7k Region ATGACTGACACCCTAGATCTAGAAATGGACGGAATTATTA overlapping CAGAGCAGCGCCTGCTAGAAAGACGCAGGGCAGCGGCCG with E3 AGCAACAGCGCATGAATCAAGAGCTCCAAGACATGGTTAA 14.5k ORF in CTTGCACCAGTGCAAAAGGGGTATCTTTTGTCTGGTAAAGC bold AGGCCAAAGTCACCTACGACAGTAATACCACCGGACACCG CCTTAGCTACAAGTTGCCAACCAAGCGTCAGAAATTGGTG GTCATGGTGGGAGAAAAGCCCATTACCATAACTCAGCACT CGGTAGAAACCGAAGGCTGCATTCACTCACCTTGTCAAGG ACCTGAGGATCTCTGCACCCTTATTAAGACCCTGTGCGGTC TCAAAGATCTTATTCCCTTTAACTAA 83 E3 14.5k Region ATGAAATTTACTGTGACTTTTCTGCTGATTATTTGCACCCTA overlapping TCTGCGTTTTGTTCCCCGACCTCCAAGCCTCAAAGACATAT with E3 ATCATGCAGATTCACTCGTATATGGAATATTCCAAGTTGCT 14.7k in bold ACAATGAAAAAAGCGATCTTTCCGAAGCCTGGTTATATGC AATCATCTCTGTTATGGTGTTCTGCAGTACCATCTTAGCCC TAGCTATATATCCCTACCTTGACATTGGCTGGAACGCAATA GATGCCATGAACCACCCAACTTTCCCCGCGCCCGCTATGCT TCCACTGCAACAAGTTGTTGCCGGCGGCTTTGTCCCAGCCA ATCAGCCTCGCCCACCTTCTCCCACCCCCACTGAAATCAGC TACTTTAATCTAACAGGAGGAGATGACTGA 84 E3 10.4K ATGATTCCTCGAGTTTTTATATTACTGACCCTTGTTGCGCTT TTTTTGTGCGTGCTCCACATTGGCTGCGGTTTCTCACATCG AAGTAGACTGCATTCCAGCCTTCACAGTCTATTTGCTTTAC GGATTTGTCACCCTCACGCTCATCTGCAGCCTCATCACTGT GGTCATCGCCTTTATCCAGTGCATTGA 85 E3 10.5k ATGACCAACACAACCAACGCGGCCGCCGCTACCGGACTTA (ADP) CATCTACCACAAATACACCCCAAGTTTCTGCCTTTGTCAAT AACTGGGATAACTTGGGCATGTGGTGGTTCTCCATAGCGCT TATGTTTGTATGCCTTATTATTATGTGGCTCATCTGCTGCCT AAAGCGCAAACGCGCCCGACCACCCATCTATAGTCCCATC ATTGTGCTACACCCAAACAATGATGGAATCCATAGATTGG ACGGACTGAAACACATGTTCTTTTCTCTTACAGTATGA 86 TRZ000 intact Region ATGATTAGGTACATAATCCTAGGTTTACTCACCCTTGCGTC E3 gp19k ORF overlapping AGCCCACGGTACCACCCAAAAGGTGGATTTTAAGGAGCCA with E3 GCCTGTAATGTTACATTCGCAGCTGAAGCTAATGAGTGCAC gp19k ORF CACTCTTATAAAATGCACCACAGAACATGAAAAGCTGCTT in bold ATTCGCCACAAAAACAAAATTGGCAAGTATGCTGTTTATG CTATTTGGCAGCCAGGTGACACTACAGAGTATAATGTTAC AGTTTTCCAGGGTAAAAGTCATAAAACTTTTATGTATACTT TTCCATTTTATGAAATGTGCGACATTACCATGTACATGAGC AAACAGTATAAGTTGTGGCCCCCACAAAATTGTGTGGAAA ACACTGGCACTTTCTGCTGCACTGCTATGCTAATTACAGTG CTCGCTTTGGTCTGTACCCTACTCTATATTAAATACAAAAG CAGACGCAGCTTTATTGAGGAAAAGAAAATGCCTTAA 87 TRZ000 intact Region ATGAACAATTCAAGCAACTCTACGGGCTATTCTAATTCAGG E3 7.1K overlapping TTTCTCTAGAATCGGGGTTGGGGTTATTCTCTGTCTTGTGAT with E3 TCTCTTTATTCTTATACTAACGCTTCTCTGCCTAAGGCTCGC gp19k ORF CGCCTGCTGTGTGCACATTTGCATTTATTGTCAGCTTTTTAA in bold ACGCTGGGGTCGCCACCCAAGATGA 88 TRZ200 intact ATGTTAAGTGGAGAGGCAGAGCAACTGCGCCTGAAACACC E3 12.5k TGGTCCACTGTCGCCGCCACAAGTGCTTTGCCCGCGACTCC GGTGAGTTTTGCTACTTTGAATTGCCCGAGGATCATATCGA GGGCCCGGCGCACGGCGTCCGGCTTACCGCCCAGGGAGAG CTTGCCCGTAGCCTGATTCGGGAGTTTACCCAGCGCCCCCT GCTAGTTGAGCGGGACAGGGGACCCTGTGTTCTCACTGTG ATTTGCAACTGTCCTAACCCTGGATTACATCAAGATCTTTG TTGCCATCTCTGTGCTGAGTATAATAAATACAGAAATTAA 89 TRZ000 intact CACCCTAGATCTAGAAATGGACGGAATTATTACAGAGCAG E3 14.7k ORF CGCCTGCTAGAAAGACGCAGGGCAGCGGCCGAGCAACAG CGCATGAATCAAGAGCTCCAAGACATGGTTAACTTGCACC AGTGCAAAAGGGGTATCTTTTGTCTGGTAAAGCAGGCCAA AGTCACCTACGACAGTAATACCACCGGACACCGCCTTAGC TACAAGTTGCCAACCAAGCGTCAGAAATTGGTGGTCATGG TGGGAGAAAAGCCCATTACCATAACTCAGCACTCGGTAGA AACCGAAGGCTGCATTCACTCACCTTGTCAAGGACCTGAG GATCTCTGCACCCTTATTAAGACCCTGTGCGGTCTCAAAGA TCTTATTCCCTTTAACTAA 90 TRZ200 E3 AGACGCCCAGGCCGAAGTTCAGATGACTAACTCAGGGGCG region (not full CAGCTTGCGGGCGGCTTTCGTCACAGGGTGCGGTCGCCCG E3-this is GGCAGGGTATAACTCACCTGACAATCAGAGGGCGAGGTAT deleted TCAGCTCAACGACGAGTCGGTGAGCTCCTCGCTTGGTCTCC version) GTCCGGACGGGACATTTCAGATCGGCGGCGCCGGCCGCTC TTCATTCACGCCTCGTCAGGCAATCCTAACTCTGCAGACCT CGTCCTCTGAGCCGCGCTCTGGAGGCATTGGAACTCTGCAA TTTATTGAGGAGTTTGTGCCATCGGTCTACTTTAACCCCTTC TCGGGACCTCCCGGCCACTATCCGGATCAATTTATTCCTAA CTTTGACGCGGTAAAGGACTCGGCGGACGGCTACGACTGA ATGTTAAGTGGAGAGGCAGAGCAACTGCGCCTGAAACACC TGGTCCACTGTCGCCGCCACAAGTGCTTTGCCCGCGACTCC GGTGAGTTTTGCTACTTTGAATTGCCCGAGGATCATATCGA GGGCCCGGCGCACGGCGTCCGGCTTACCGCCCAGGGAGAG CTTGCCCGTAGCCTGATTCGGGAGTTTACCCAGCGCCCCCT GCTAGTTGAGCGGGACAGGGGACCCTGTGTTCTCACTGTG ATTTGCAACTGTCCTAACCCTGGATTACATCAAGATCCTCT AGTTAATGTCAGGTCGCCTAAGTCGATTAACTAGAGTACCC GGGGATCTTATTCCCTTTAACTAA 91 TRZ200 GATCTTATTCCCTTTAACTAA deleted E3 14.7k partial ORF 92 TRZ200 ATGTTAAGTGGAGAGGCAGAGCAACTGCGCCTGAAACACC deleted E3 TGGTCCACTGTCGCCGCCACAAGTGCTTTGCCCGCGACTCC 12.5K partial GGTGAGTTTTGCTACTTTGAATTGCCCGAGGATCATATCGA ORF GGGCCCGGCGCACGGCGTCCGGCTTACCGCCCAGGGAGAG CTTGCCCGTAGCCTGATTCGGGAGTTTACCCAGCGCCCCCT GCTAGTTGAGCGGGACAGGGGACCCTGTGTTCTCACTGTG ATTTGCAACTGTCCTAACCCTGGATTACATCAAGAT 93 E3 Promoter AGACGCCCAGGCCGAAGTTCAGATGACTA (E3 deleted or E3 intact) 94 Full TRZ200 TTTTGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTT virus sequence TGTGACGTGGCGCGGGGCGTGGGAACGGGGCGGGTGACGT beginning at 5′ AGTAGTGTGGCGGAAGTGTGATGTTGCAAGTGTGGCGGAA ITR through 3′ CACATGTAAGCGACGGATGTGGCAAAAGTGACGTTTTTGG ITR TGTGCGCCGGTGTTTTGGGCGTAACCGAGTAAGATTTGGCC ATTTTCGCGGGAAAACTGAATAAGAGGAAGTGAAATCTGA ATAATTTTGTGTTACTCATAGCGCGTAATATTTGTCTAGGG CCGCGGGGACTTTGACCGTTTACGTGGAGACTCGCCCAGG TGTTTTTCTCAGGTGTTTTCCGCGTTCCGGGTCAAAGTTGG CGTTTTATTATTATAGTCAGCTGACGTGTAGTGTATTTATA CCCGGTGAGTTCCTCAAGAGGCCACTCTTGAGTGCCAGCG AGTAGAGTTTTCTCCTCCGAGCCGCTCCGACACCGGGACTG AAAATGAGACATATTATCTGCCACGGAGGTGTTATTACCG AAGAAATGGCCGCCAGTCTTTTGGACCAGCTGATCGAAGA GGTACTGGCTGATAATCTTCCACCTCCTAGCCATTTTGAAC CACCTACCCTTCACGAACTGTATGATTTAGACGTGACGGCC CCCGAAGATCCCAACGAGGAGGCGGTTTCGCAGATTTTTC CCGACTCTGTAATGTTGGCGGTGCAGGAAGGGATTGACTT ACTCACTTTTCCGCCGGCGCCCGGTTCTCCGGAGCCGCCTC ACCTTTCCCGGCAGCCCGAGCAGCCGGAGCAGAGAGCCTT GGGTCCGGTTTCTATGCCAAACCTTGTACCGGAGGTGATCG ATCTTACCTGCCACGAGGCTGGCTTTCCACCCAGTGACGAC GAGGATGAAGAGGGTGAGGAGTTTGTGTTAGATTATGTGG AGCACCCCGGGCACGGTTGCAGGTCTTGTCATTATCACCGG AGGAATACGGGGGACCCAGATATTATGTGTTCGCTTTGCTA TATGAGGACCTGTGGCATGTTTGTCTACAGTAAGTGAAAAT TATGGGCAGTGGGTGATAGAGTGGTGGGTTTGGTGTGGTA ATTTTTTTTTTAATTTTTACAGTTTTGTGGTTTAAAGAATTT TGTATTGTGATTTTTTTAAAAGGTCCTGTGTCTGAACCTGA GCCTGAGCCCGAGCCAGAACCGGAGCCTGCAAGACCTACC CGCCGTCCTAAAATGGCGCCTGCTATCCTGAGACGCCCGA CATCACCTGTGTCTAGAGAATGCAATAGTAGTACGGATAG CTGTGACTCCGGTCCTTCTAACACACCTCCTGAGATACACC CGGTGGTCCCGCTGTGCCCCATTAAACCAGTTGCCGTGAGA GTTGGTGGGCGTCGCCAGGCTGTGGAATGTATCGAGGACT TGCTTAACGAGCCTGGGCAACCTTTGGACTTGAGCTGTAAA CGCCCCAGGCCATAAGGTGTAAACCTGTGATTGCGTGTGT GGTTAACGCCTTTGTTTGCTGAATGAGTTGATGTAAGTTTA ATAAAGGGTGAGATAATGTTTAACTTGCATGGCGTGTTAA ATGGGGCGGGGCTTAAAGGGTATATAATGCGCCGTGGGCT AATCTTGGTTACATCTGACCTCGTCGACGCTTGGGAGTGTT TGGAAGATTTTTCTGCTGTGCGTAACTTGCTGGAACAGAGC TCTAACAGTACCTCTTGGTTTTGGAGGTTTCTGTGGGGCTC ATCCCAGGCAAAGTTAGTCTGCAGAATTAAGGAGGATTAC AAGTGGGAATTTGAAGAGCTTTTGAAATCCTGTGGTGAGC TGTTTGATTCTTTGAACTCGAGTCACCAGGCGCTTTTCCAA GAGAAGGTCATCAAGACTTTGGATTTTTCCACACCGGGGC GCGCTGCGGCTGCTGTTGCTTTTTTGAGTTTTATAAAGGAT AAATGGAGCGAAGAAACCCATCTGAGCGGGGGGTACCTGC TGGATTTTCTGGCCATGCATCTGTGGAGAGCGGTTGTGAGA CACAAGAATCGCCTGCTACTGTTGTCTTCCGTCCGCCCGGC GATAATACCGACGGAGGAGCAGCAGCAGCAGCAGGAGGA AGCCAGGCGGCGGCGGCAGGAGCAGAGCCCATGGAACCC GAGAGCCGGCCTGGACCCTCGGGAATGAATGTTGTACAGG TGGCTGAACTGTATCCAGAACTGAGACGCATTTTGACAATT ACAGAGGATGGGCAGGGGCTAAAGGGGGTAAAGAGGGAG CGGGGGGCTTGTGAGGCTACAGAGGAGGCTAGGAATCTAG CTTTTAGCTTAATGACCAGACACCGTCCTGAGTGTATTACT TTTCAACAGATCAAGGATAATTGCGCTAATGAGCTTGATCT GCTGGCGCAGAAGTATTCCATAGAGCAGCTGACCACTTAC TGGCTGCAGCCAGGGGATGATTTTGAGGAGGCTATTAGGG TATATGCAAAGGTGGCACTTAGGCCAGATTGCAAGTACAA GATCAGCAAACTTGTAAATATCAGGAATTGTTGCTACATTT CTGGGAACGGGGCCGAGGTGGAGATAGATACGGAGGATA GGGTGGCCTTTAGATGTAGCATGATAAATATGTGGCCGGG GGTGCTTGGCATGGACGGGGTGGTTATTATGAATGTAAGG TTTACTGGCCCCAATTTTAGCGGTACGGTTTTCCTGGCCAA TACCAACCTTATCCTACACGGTGTAAGCTTCTATGGGTTTA ACAATACCTGTGTGGAAGCCTGGACCGATGTAAGGGTTCG GGGCTGTGCCTTTTACTGCTGCTGGAAGGGGGTGGTGTGTC GCCCCAAAAGCAGGGCTTCAATTAAGAAATGCCTCTTTGA AAGGTGTACCTTGGGTATCCTGTCTGAGGGTAACTCCAGG GTGCGCCACAATGTGGCCTCCGACTGTGGTTGCTTCATGCT AGTGAAAAGCGTGGCTGTGATTAAGCATAACATGGTATGT GGCAACTGCGAGGACAGGGCCTCTCAGATGCTGACCTGCT CGGACGGCAACTGTCACCTGCTGAAGACCATTCACGTAGC CAGCCACTCTCGCAAGGCCTGGCCAGTGTTTGAGCATAAC ATACTGACCCGCTGTTCCTTGCATTTGGGTAACAGGAGGGG GGTGTTCCTACCTTACCAATGCAATTTGAGTCACACTAAGA TATTGCTTGAGCCCGAGAGCATGTCCAAGGTGAACCTGAA CGGGGTGTTTGACATGACCATGAAGATCTGGAAGGTGCTG AGGTACGATGAGACCCGCACCAGGTGCAGACCCTGCGAGT GTGGCGGTAAACATATTAGGAACCAGCCTGTGATGCTGGA TGTGACCGAGGAGCTGAGGCCCGATCACTTGGTGCTGGCC TGCACCCGCGCTGAGTTTGGCTCTAGCGATGAAGATACAG ATTGAGGTACTGAAATGTGTGGGCGTGGCTTAAGGGTGGG AAAGAATATATAAGGTGGGGGTCTTATGTAGTTTTGTATCT GTTTTGCAGCAGCCGCCGCCGCCATGAGCACCAACTCGTTT GATGGAAGCATTGTGAGCTCATATTTGACAACGCGCATGC CCCCATGGGCCGGGGTGCGTCAGAATGTGATGGGCTCCAG CATTGATGGTCGCCCCGTCCTGCCCGCAAACTCTACTACCT TGACCTACGAGACCGTGTCTGGAACGCCGTTGGAGACTGC AGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCACCGCCCGC GGGATTGTGACTGACTTTGCTTTCCTGAGCCCGCTTGCAAG CAGTGCAGCTTCCCGTTCATCCGCCCGCGATGACAAGTTGA CGGCTCTTTTGGCACAATTGGATTCTTTGACCCGGGAACTT AATGTCGTTTCTCAGCAGCTGTTGGATCTGCGCCAGCAGGT TTCTGCCCTGAAGGCTTCCTCCCCTCCCAATGCGGTTTAAA ACATAAATAAAAAACCAGACTCTGTTTGGATTTGGATCAA GCAAGTGTCTTGCTGTCTTTATTTAGGGGTTTTGCGCGCGC GGTAGGCCCGGGACCAGCGGTCTCGGTCGTTGAGGGTCCT GTGTATTTTTTCCAGGACGTGGTAAAGGTGACTCTGGATGT TCAGATACATGGGCATAAGCCCGTCTCTGGGGTGGAGGTA GCACCACTGCAGAGCTTCATGCTGCGGGGTGGTGTTGTAG ATGATCCAGTCGTAGCAGGAGCGCTGGGCGTGGTGCCTAA AAATGTCTTTCAGTAGCAAGCTGATTGCCAGGGGCAGGCC CTTGGTGTAAGTGTTTACAAAGCGGTTAAGCTGGGATGGG TGCATACGTGGGGATATGAGATGCATCTTGGACTGTATTTT TAGGTTGGCTATGTTCCCAGCCATATCCCTCCGGGGATTCA TGTTGTGCAGAACCACCAGCACAGTGTATCCGGTGCACTTG GGAAATTTGTCATGTAGCTTAGAAGGAAATGCGTGGAAGA ACTTGGAGACGCCCTTGTGACCTCCAAGATTTTCCATGCAT TCGTCCATAATGATGGCAATGGGCCCACGGGCGGCGGCCT GGGCGAAGATATTTCTGGGATCACTAACGTCATAGTTGTGT TCCAGGATGAGATCGTCATAGGCCATTTTTACAAAGCGCG GGCGGAGGGTGCCAGACTGCGGTATAATGGTTCCATCCGG CCCAGGGGCGTAGTTACCCTCACAGATTTGCATTTCCCACG CTTTGAGTTCAGATGGGGGGATCATGTCTACCTGCGGGGC GATGAAGAAAACGGTTTCCGGGGTAGGGGAGATCAGCTGG GAAGAAAGCAGGTTCCTGAGCAGCTGCGACTTACCGCAGC CGGTGGGCCCGTAAATCACACCTATTACCGGCTGCAACTG GTAGTTAAGAGAGCTGCAGCTGCCGTCATCCCTGAGCAGG GGGGCCACTTCGTTAAGCATGTCCCTGACTCGCATGTTTTC CCTGACCAAATCCGCCAGAAGGCGCTCGCCGCCCAGCGAT AGCAGTTCTTGCAAGGAAGCAAAGTTTTTCAACGGTTTGA GACCGTCCGCCGTAGGCATGCTTTTGAGCGTTTGACCAAGC AGTTCCAGGCGGTCCCACAGCTCGGTCACCTGCTCTACGGC ATCTCGATCCAGCATATCTCCTCGTTTCGCGGGTTGGGGCG GCTTTCGCTGTACGGCAGTAGTCGGTGCTCGTCCAGACGGG CCAGGGTCATGTCTTTCCACGGGCGCAGGGTCCTCGTCAGC GTAGTCTGGGTCACGGTGAAGGGGTGCGCTCCGGGCTGCG CGCTGGCCAGGGTGCGCTTGAGGCTGGTCCTGCTGGTGCTG AAGCGCTGCCGGTCTTCGCCCTGCGCGTCGGCCAGGTAGC ATTTGACCATGGTGTCATAGTCCAGCCCCTCCGCGGCGTGG CCCTTGGCGCGCAGCTTGCCCTTGGAGGAGGCGCCGCACG AGGGGCAGTGCAGACTTTTGAGGGCGTAGAGCTTGGGCGC GAGAAATACCGATTCCGGGGAGTAGGCATCCGCGCCGCAG GCCCCGCAGACGGTCTCGCATTCCACGAGCCAGGTGAGCT CTGGCCGTTCGGGGTCAAAAACCAGGTTTCCCCCATGCTTT TTGATGCGTTTCTTACCTCTGGTTTCCATGAGCCGGTGTCC ACGCTCGGTGACGAAAAGGCTGTCCGTGTCCCCGTATACA GACTTGAGAGGCCTGTCCTCGAGCGGTGTTCCGCGGTCCTC CTCGTATAGAAACTCGGACCACTCTGAGACAAAGGCTCGC GTCCAGGCCAGCACGAAGGAGGCTAAGTGGGAGGGGTAG CGGTCGTTGTCCACTAGGGGGTCCACTCGCTCCAGGGTGTG AAGACACATGTCGCCCTCTTCGGCATCAAGGAAGGTGATT GGTTTGTAGGTGTAGGCCACGTGACCGGGTGTTCCTGAAG GGGGGCTATAAAAGGGGGTGGGGGCGCGTTCGTCCTCACT CTCTTCCGCATCGCTGTCTGCGAGGGCCAGCTGTTGGGGTG AGTACTCCCTCTGAAAAGCGGGCATGACTTCTGCGCTAAG ATTGTCAGTTTCCAAAAACGAGGAGGATTTGATATTCACCT GGCCCGCGGTGATGCCTTTGAGGGTGGCCGCATCCATCTG GTCAGAAAAGACAATCTTTTTGTTGTCAAGCTTGGTGGCAA ACGACCCGTAGAGGGCGTTGGACAGCAACTTGGCGATGGA GCGCAGGGTTTGGTTTTTGTCGCGATCGGCGCGCTCCTTGG CCGCGATGTTTAGCTGCACGTATTCGCGCGCAACGCACCGC CATTCGGGAAAGACGGTGGTGCGCTCGTCGGGCACCAGGT GCACGCGCCAACCGCGGTTGTGCAGGGTGACAAGGTCAAC GCTGGTGGCTACCTCTCCGCGTAGGCGCTCGTTGGTCCAGC AGAGGCGGCCGCCCTTGCGCGAGCAGAATGGCGGTAGGGG GTCTAGCTGCGTCTCGTCCGGGGGGTCTGCGTCCACGGTAA AGACCCCGGGCAGCAGGCGCGCGTCGAAGTAGTCTATCTT GCATCCTTGCAAGTCTAGCGCCTGCTGCCATGCGCGGGCG GCAAGCGCGCGCTCGTATGGGTTGAGTGGGGGACCCCATG GCATGGGGTGGGTGAGCGCGGAGGCGTACATGCCGCAAAT GTCGTAAACGTAGAGGGGCTCTCTGAGTATTCCAAGATAT GTAGGGTAGCATCTTCCACCGCGGATGCTGGCGCGCACGT AATCGTATAGTTCGTGCGAGGGAGCGAGGAGGTCGGGACC GAGGTTGCTACGGGCGGGCTGCTCTGCTCGGAAGACTATC TGCCTGAAGATGGCATGTGAGTTGGATGATATGGTTGGAC GCTGGAAGACGTTGAAGCTGGCGTCTGTGAGACCTACCGC GTCACGCACGAAGGAGGCGTAGGAGTCGCGCAGCTTGTTG ACCAGCTCGGCGGTGACCTGCACGTCTAGGGCGCAGTAGT CCAGGGTTTCCTTGATGATGTCATACTTATCCTGTCCCTTTT TTTTCCACAGCTCGCGGTTGAGGACAAACTCTTCGCGGTCT TTCCAGTACTCTTGGATCGGAAACCCGTCGGCCTCCGAACG GTAAGAGCCTAGCATGTAGAACTGGTTGACGGCCTGGTAG GCGCAGCATCCCTTTTCTACGGGTAGCGCGTATGCCTGCGC GGCCTTCCGGAGCGAGGTGTGGGTGAGCGCAAAGGTGTCC CTGACCATGACTTTGAGGTACTGGTATTTGAAGTCAGTGTC GTCGCATCCGCCCTGCTCCCAGAGCAAAAAGTCCGTGCGC TTTTTGGAACGCGGATTTGGCAGGGCGAAGGTGACATCGT TGAAGAGTATCTTTCCCGCGCGAGGCATAAAGTTGCGTGT GATGCGGAAGGGTCCCGGCACCTCGGAACGGTTGTTAATT ACCTGGGCGGCGAGCACGATCTCGTCAAAGCCGTTGATGT TGTGGCCCACAATGTAAAGTTCCAAGAAGCGCGGGATGCC CTTGATGGAAGGCAATTTTTTAAGTTCCTCGTAGGTGAGCT CTTCAGGGGAGCTGAGCCCGTGCTCTGAAAGGGCCCAGTC TGCAAGATGAGGGTTGGAAGCGACGAATGAGCTCCACAGG TCACGGGCCATTAGCATTTGCAGGTGGTCGCGAAAGGTCC TAAACTGGCGACCTATGGCCATTTTTTCTGGGGTGATGCAG TAGAAGGTAAGCGGGTCTTGTTCCCAGCGGTCCCATCCAA GGTTCGCGGCTAGGTCTCGCGCGGCAGTCACTAGAGGCTC ATCTCCGCCGAACTTCATGACCAGCATGAAGGGCACGAGC TGCTTCCCAAAGGCCCCCATCCAAGTATAGGTCTCTACATC GTAGGTGACAAAGAGACGCTCGGTGCGAGGATGCGAGCCG ATCGGGAAGAACTGGATCTCCCGCCACCAATTGGAGGAGT GGCTATTGATGTGGTGAAAGTAGAAGTCCCTGCGACGGGC CGAACACTCGTGCTGGCTTTTGTAAAAACGTGCGCAGTACT GGCAGCGGTGCACGGGCTGTACATCCTGCACGAGGTTGAC CTGACGACCGCGCACAAGGAAGCAGAGTGGGAATTTGAGC CCCTCGCCTGGCGGGTTTGGCTGGTGGTCTTCTACTTCGGC TGCTTGTCCTTGACCGTCTGGCTGCTCGAGGGGAGTTACGG TGGATCGGACCACCACGCCGCGCGAGCCCAAAGTCCAGAT GTCCGCGCGCGGCGGTCGGAGCTTGATGACAACATCGCGC AGATGGGAGCTGTCCATGGTCTGGAGCTCCCGCGGCGTCA GGTCAGGCGGGAGCTCCTGCAGGTTTACCTCGCATAGACG GGTCAGGGCGCGGGCTAGATCCAGGTGATACCTAATTTCC AGGGGCTGGTTGGTGGCGGCGTCGATGGCTTGCAAGAGGC CGCATCCCCGCGGCGCGACTACGGTACCGCGCGGCGGGCG GTGGGCCGCGGGGGTGTCCTTGGATGATGCATCTAAAAGC GGTGACGCGGGCGAGCCCCCGGAGGTAGGGGGGGCTCCG GACCCGCCGGGAGAGGGGGCAGGGGCACGTCGGCGCCGC GCGCGGGCAGGAGCTGGTGCTGCGCGCGTAGGTTGCTGGC GAACGCGACGACGCGGCGGTTGATCTCCTGAATCTGGCGC CTCTGCGTGAAGACGACGGGCCCGGTGAGCTTGAACCTGA AAGAGAGTTCGACAGAATCAATTTCGGTGTCGTTGACGGC GGCCTGGCGCAAAATCTCCTGCACGTCTCCTGAGTTGTCTT GATAGGCGATCTCGGCCATGAACTGCTCGATCTCTTCCTCC TGGAGATCTCCGCGTCCGGCTCGCTCCACGGTGGCGGCGA GGTCGTTGGAAATGCGGGCCATGAGCTGCGAGAAGGCGTT GAGGCCTCCCTCGTTCCAGACGCGGCTGTAGACCACGCCC CCTTCGGCATCGCGGGCGCGCATGACCACCTGCGCGAGAT TGAGCTCCACGTGCCGGGCGAAGACGGCGTAGTTTCGCAG GCGCTGAAAGAGGTAGTTGAGGGTGGTGGCGGTGTGTTCT GCCACGAAGAAGTACATAACCCAGCGTCGCAACGTGGATT CGTTGATATCCCCCAAGGCCTCAAGGCGCTCCATGGCCTCG TAGAAGTCCACGGCGAAGTTGAAAAACTGGGAGTTGCGCG CCGACACGGTTAACTCCTCCTCCAGAAGACGGATGAGCTC GGCGACAGTGTCGCGCACCTCGCGCTCAAAGGCTACAGGG GCCTCTTCTTCTTCTTCAATCTCCTCTTCCATAAGGGCCTCC CCTTCTTCTTCTTCTGGCGGCGGTGGGGGAGGGGGGACAC GGCGGCGACGACGGCGCACCGGGAGGCGGTCGACAAAGC GCTCGATCATCTCCCCGCGGCGACGGCGCATGGTCTCGGTG ACGGCGCGGCCGTTCTCGCGGGGGCGCAGTTGGAAGACGC CGCCCGTCATGTCCCGGTTATGGGTTGGCGGGGGGCTGCC ATGCGGCAGGGATACGGCGCTAACGATGCATCTCAACAAT TGTTGTGTAGGTACTCCGCCGCCGAGGGACCTGAGCGAGT CCGCATCGACCGGATCGGAAAACCTCTCGAGAAAGGCGTC TAACCAGTCACAGTCGCAAGGTAGGCTGAGCACCGTGGCG GGCGGCAGCGGGCGGCGGTCGGGGTTGTTTCTGGCGGAGG TGCTGCTGATGATGTAATTAAAGTAGGCGGTCTTGAGACG GCGGATGGTCGACAGAAGCACCATGTCCTTGGGTCCGGCC TGCTGAATGCGCAGGCGGTCGGCCATGCCCCAGGCTTCGTT TTGACATCGGCGCAGGTCTTTGTAGTAGTCTTGCATGAGCC TTTCTACCGGCACTTCTTCTTCTCCTTCCTCTTGTCCTGCAT CTCTTGCATCTATCGCTGCGGCGGCGGCGGAGTTTGGCCGT AGGTGGCGCCCTCTTCCTCCCATGCGTGTGACCCCGAAGCC CCTCATCGGCTGAAGCAGGGCTAGGTCGGCGACAACGCGC TCGGCTAATATGGCCTGCTGCACCTGCGTGAGGGTAGACT GGAAGTCATCCATGTCCACAAAGCGGTGGTATGCGCCCGT GTTGATGGTGTAAGTGCAGTTGGCCATAACGGACCAGTTA ACGGTCTGGTGACCCGGCTGCGAGAGCTCGGTGTACCTGA GACGCGAGTAAGCCCTCGAGTCAAATACGTAGTCGTTGCA AGTCCGCACCAGGTACTGGTATCCCACCAAAAAGTGCGGC GGCGGCTGGCGGTAGAGGGGCCAGCGTAGGGTGGCCGGG GCTCCGGGGGCGAGATCTTCCAACATAAGGCGATGATATC CGTAGATGTACCTGGACATCCAGGTGATGCCGGCGGCGGT GGTGGAGGCGCGCGGAAAGTCGCGGACGCGGTTCCAGATG TTGCGCAGCGGCAAAAAGTGCTCCATGGTCGGGACGCTCT GGCCGGTCAGGCGCGCGCAATCGTTGACGCTCTAGCGTGC AAAAGGAGAGCCTGTAAGCGGGCACTCTTCCGTGGTCTGG TGGATAAATTCGCAAGGGTATCATGGCGGACGACCGGGGT TCGAGCCCCGTATCCGGCCGTCCGCCGTGATCCATGCGGTT ACCGCCCGCGTGTCGAACCCAGGTGTGCGACGTCAGACAA CGGGGGAGTGCTCCTTTTGGCTTCCTTCCAGGCGCGGCGGC TGCTGCGCTAGCTTTTTTGGCCACTGGCCGCGCGCAGCGTA AGCGGTTAGGCTGGAAAGCGAAAGCATTAAGTGGCTCGCT CCCTGTAGCCGGAGGGTTATTTTCCAAGGGTTGAGTCGCGG GACCCCCGGTTCGAGTCTCGGACCGGCCGGACTGCGGCGA ACGGGGGTTTGCCTCCCCGTCATGCAAGACCCCGCTTGCAA ATTCCTCCGGAAACAGGGACGAGCCCCTTTTTTGCTTTTCC CAGATGCATCCGGTGCTGCGGCAGATGCGCCCCCCTCCTCA GCAGCGGCAAGAGCAAGAGCAGCGGCAGACATGCAGGGC ACCCTCCCCTCCTCCTACCGCGTCAGGAGGGGCGACATCCG CGGTTGACGCGGCAGCAGATGGTGATTACGAACCCCCGCG GCGCCGGGCCCGGCACTACCTGGACTTGGAGGAGGGCGAG GGCCTGGCGCGGCTAGGAGCGCCCTCTCCTGAGCGGCACC CAAGGGTGCAGCTGAAGCGTGATACGCGTGAGGCGTACGT GCCGCGGCAGAACCTGTTTCGCGACCGCGAGGGAGAGGAG CCCGAGGAGATGCGGGATCGAAAGTTCCACGCAGGGCGCG AGCTGCGGCATGGCCTGAATCGCGAGCGGTTGCTGCGCGA GGAGGACTTTGAGCCCGACGCGCGAACCGGGATTAGTCCC GCGCGCGCACACGTGGCGGCCGCCGACCTGGTAACCGCAT ACGAGCAGACGGTGAACCAGGAGATTAACTTTCAAAAAAG CTTTAACAACCACGTGCGTACGCTTGTGGCGCGCGAGGAG GTGGCTATAGGACTGATGCATCTGTGGGACTTTGTAAGCGC GCTGGAGCAAAACCCAAATAGCAAGCCGCTCATGGCGCAG CTGTTCCTTATAGTGCAGCACAGCAGGGACAACGAGGCAT TCAGGGATGCGCTGCTAAACATAGTAGAGCCCGAGGGCCG CTGGCTGCTCGATTTGATAAACATCCTGCAGAGCATAGTGG TGCAGGAGCGCAGCTTGAGCCTGGCTGACAAGGTGGCCGC CATCAACTATTCCATGCTTAGCCTGGGCAAGTTTTACGCCC GCAAGATATACCATACCCCTTACGTTCCCATAGACAAGGA GGTAAAGATCGAGGGGTTCTACATGCGCATGGCGCTGAAG GTGCTTACCTTGAGCGACGACCTGGGCGTTTATCGCAACGA GCGCATCCACAAGGCCGTGAGCGTGAGCCGGCGGCGCGAG CTCAGCGACCGCGAGCTGATGCACAGCCTGCAAAGGGCCC TGGCTGGCACGGGCAGCGGCGATAGAGAGGCCGAGTCCTA CTTTGACGCGGGCGCTGACCTGCGCTGGGCCCCAAGCCGA CGCGCCCTGGAGGCAGCTGGGGCCGGACCTGGGCTGGCGG TGGCACCCGCGCGCGCTGGCAACGTCGGCGGCGTGGAGGA ATATGACGAGGACGATGAGTACGAGCCAGAGGACGGCGA GTACTAAGCGGTGATGTTTCTGATCAGATGATGCAAGACG CAACGGACCCGGCGGTGCGGGCGGCGCTGCAGAGCCAGCC GTCCGGCCTTAACTCCACGGACGACTGGCGCCAGGTCATG GACCGCATCATGTCGCTGACTGCGCGCAATCCTGACGCGTT CCGGCAGCAGCCGCAGGCCAACCGGCTCTCCGCAATTCTG GAAGCGGTGGTCCCGGCGCGCGCAAACCCCACGCACGAGA AGGTGCTGGCGATCGTAAACGCGCTGGCCGAAAACAGGGC CATCCGGCCCGACGAGGCCGGCCTGGTCTACGACGCGCTG CTTCAGCGCGTGGCTCGTTACAACAGCGGCAACGTGCAGA CCAACCTGGACCGGCTGGTGGGGGATGTGCGCGAGGCCGT GGCGCAGCGTGAGCGCGCGCAGCAGCAGGGCAACCTGGG CTCCATGGTTGCACTAAACGCCTTCCTGAGTACACAGCCCG CCAACGTGCCGCGGGGACAGGAGGACTACACCAACTTTGT GAGCGCACTGCGGCTAATGGTGACTGAGACACCGCAAAGT GAGGTGTACCAGTCTGGGCCAGACTATTTTTTCCAGACCAG TAGACAAGGCCTGCAGACCGTAAACCTGAGCCAGGCTTTC AAAAACTTGCAGGGGCTGTGGGGGGTGCGGGCTCCCACAG GCGACCGCGCGACCGTGTCTAGCTTGCTGACGCCCAACTC GCGCCTGTTGCTGCTGCTAATAGCGCCCTTCACGGACAGTG GCAGCGTGTCCCGGGACACATACCTAGGTCACTTGCTGAC ACTGTACCGCGAGGCCATAGGTCAGGCGCATGTGGACGAG CATACTTTCCAGGAGATTACAAGTGTCAGCCGCGCGCTGG GGCAGGAGGACACGGGCAGCCTGGAGGCAACCCTAAACT ACCTGCTGACCAACCGGCGGCAGAAGATCCCCTCGTTGCA CAGTTTAAACAGCGAGGAGGAGCGCATTTTGCGCTACGTG CAGCAGAGCGTGAGCCTTAACCTGATGCGCGACGGGGTAA CGCCCAGCGTGGCGCTGGACATGACCGCGCGCAACATGGA ACCGGGCATGTATGCCTCAAACCGGCCGTTTATCAACCGCC TAATGGACTACTTGCATCGCGCGGCCGCCGTGAACCCCGA GTATTTCACCAATGCCATCTTGAACCCGCACTGGCTACCGC CCCCTGGTTTCTACACCGGGGGATTCGAGGTGCCCGAGGG TAACGATGGATTCCTCTGGGACGACATAGACGACAGCGTG TTTTCCCCGCAACCGCAGACCCTGCTAGAGTTGCAACAGCG CGAGCAGGCAGAGGCGGCGCTGCGAAAGGAAAGCTTCCG CAGGCCAAGCAGCTTGTCCGATCTAGGCGCTGCGGCCCCG CGGTCAGATGCTAGTAGCCCATTTCCAAGCTTGATAGGGTC TCTTACCAGCACTCGCACCACCCGCCCGCGCCTGCTGGGCG AGGAGGAGTACCTAAACAACTCGCTGCTGCAGCCGCAGCG CGAAAAAAACCTGCCTCCGGCATTTCCCAACAACGGGATA GAGAGCCTAGTGGACAAGATGAGTAGATGGAAGACGTAC GCGCAGGAGCACAGGGACGTGCCAGGCCCGCGCCCGCCCA CCCGTCGTCAAAGGCACGACCGTCAGCGGGGTCTGGTGTG GGAGGACGATGACTCGGCAGACGACAGCAGCGTCCTGGAT TTGGGAGGGAGTGGCAACCCGTTTGCGCACCTTCGCCCCA GGCTGGGGAGAATGTTTTAAAAAAAAAAAAGCATGATGCA AAATAAAAAACTCACCAAGGCCATGGCACCGAGCGTTGGT TTTCTTGTATTCCCCTTAGTATGCGGCGCGCGGCGATGTAT GAGGAAGGTCCTCCTCCCTCCTACGAGAGTGTGGTGAGCG CGGCGCCAGTGGCGGCGGCGCTGGGTTCTCCCTTCGATGCT CCCCTGGACCCGCCGTTTGTGCCTCCGCGGTACCTGCGGCC TACCGGGGGGAGAAACAGCATCCGTTACTCTGAGTTGGCA CCCCTATTCGACACCACCCGTGTGTACCTGGTGGACAACAA GTCAACGGATGTGGCATCCCTGAACTACCAGAACGACCAC AGCAACTTTCTGACCACGGTCATTCAAAACAATGACTACA GCCCGGGGGAGGCAAGCACACAGACCATCAATCTTGACGA CCGGTCGCACTGGGGCGGCGACCTGAAAACCATCCTGCAT ACCAACATGCCAAATGTGAACGAGTTCATGTTTACCAATA AGTTTAAGGCGCGGGTGATGGTGTCGCGCTTGCCTACTAA GGACAATCAGGTGGAGCTGAAATACGAGTGGGTGGAGTTC ACGCTGCCCGAGGGCAACTACTCCGAGACCATGACCATAG ACCTTATGAACAACGCGATCGTGGAGCACTACTTGAAAGT GGGCAGACAGAACGGGGTTCTGGAAAGCGACATCGGGGT AAAGTTTGACACCCGCAACTTCAGACTGGGGTTTGACCCC GTCACTGGTCTTGTCATGCCTGGGGTATATACAAACGAAGC CTTCCATCCAGACATCATTTTGCTGCCAGGATGCGGGGTGG ACTTCACCCACAGCCGCCTGAGCAACTTGTTGGGCATCCGC AAGCGGCAACCCTTCCAGGAGGGCTTTAGGATCACCTACG ATGATCTGGAGGGTGGTAACATTCCCGCACTGTTGGATGTG GACGCCTACCAGGCGAGCTTGAAAGATGACACCGAACAGG GCGGGGGTGGCGCAGGCGGCAGCAACAGCAGTGGCAGCG GCGCGGAAGAGAACTCCAACGCGGCAGCCGCGGCAATGC AGCCGGTGGAGGACATGAACGATCATGCCATTCGCGGCGA CACCTTTGCCACACGGGCTGAGGAGAAGCGCGCTGAGGCC GAAGCAGCGGCCGAAGCTGCCGCCCCCGCTGCGCAACCCG AGGTCGAGAAGCCTCAGAAGAAACCGGTGATCAAACCCCT GACAGAGGACAGCAAGAAACGCAGTTACAACCTAATAAG CAATGACAGCACCTTCACCCAGTACCGCAGCTGGTACCTTG CATACAACTACGGCGACCCTCAGACCGGAATCCGCTCATG GACCCTGCTTTGCACTCCTGACGTAACCTGCGGCTCGGAGC AGGTCTACTGGTCGTTGCCAGACATGATGCAAGACCCCGT GACCTTCCGCTCCACGCGCCAGATCAGCAACTTTCCGGTGG TGGGCGCCGAGCTGTTGCCCGTGCACTCCAAGAGCTTCTAC AACGACCAGGCCGTCTACTCCCAACTCATCCGCCAGTTTAC CTCTCTGACCCACGTGTTCAATCGCTTTCCCGAGAACCAGA TTTTGGCGCGCCCGCCAGCCCCCACCATCACCACCGTCAGT GAAAACGTTCCTGCTCTCACAGATCACGGGACGCTACCGC TGCGCAACAGCATCGGAGGAGTCCAGCGAGTGACCATTAC TGACGCCAGACGCCGCACCTGCCCCTACGTTTACAAGGCC CTGGGCATAGTCTCGCCGCGCGTCCTATCGAGCCGCACTTT TTGAGCAAGCATGTCCATCCTTATATCGCCCAGCAATAACA CAGGCTGGGGCCTGCGCTTCCCAAGCAAGATGTTTGGCGG GGCCAAGAAGCGCTCCGACCAACACCCAGTGCGCGTGCGC GGGCACTACCGCGCGCCCTGGGGCGCGCACAAACGCGGCC GCACTGGGCGCACCACCGTCGATGACGCCATCGACGCGGT GGTGGAGGAGGCGCGCAACTACACGCCCACGCCGCCACCA GTGTCCACAGTGGACGCGGCCATTCAGACCGTGGTGCGCG GAGCCCGGCGCTATGCTAAAATGAAGAGACGGCGGAGGC GCGTAGCACGTCGCCACCGCCGCCGACCCGGCACTGCCGC CCAACGCGCGGCGGCGGCCCTGCTTAACCGCGCACGTCGC ACCGGCCGACGGGCGGCCATGCGGGCCGCTCGAAGGCTGG CCGCGGGTATTGTCACTGTGCCCCCCAGGTCCAGGCGACG AGCGGCCGCCGCAGCAGCCGCGGCCATTAGTGCTATGACT CAGGGTCGCAGGGGCAACGTGTATTGGGTGCGCGACTCGG TTAGCGGCCTGCGCGTGCCCGTGCGCACCCGCCCCCCGCGC AACTAGATTGCAAGAAAAAACTACTTAGACTCGTACTGTT GTATGTATCCAGCGGCGGCGGCGCGCAACGAAGCTATGTC CAAGCGCAAAATCAAAGAAGAGATGCTCCAGGTCATCGCG CCGGAGATCTATGGCCCCCCGAAGAAGGAAGAGCAGGATT ACAAGCCCCGAAAGCTAAAGCGGGTCAAAAAGAAAAAGA AAGATGATGATGATGAACTTGACGACGAGGTGGAACTGCT GCACGCTACCGCGCCCAGGCGACGGGTACAGTGGAAAGGT CGACGCGTAAAACGTGTTTTGCGACCCGGCACCACCGTAG TCTTTACGCCCGGTGAGCGCTCCACCCGCACCTACAAGCGC GTGTATGATGAGGTGTACGGCGACGAGGACCTGCTTGAGC AGGCCAACGAGCGCCTCGGGGAGTTTGCCTACGGAAAGCG GCATAAGGACATGCTGGCGTTGCCGCTGGACGAGGGCAAC CCAACACCTAGCCTAAAGCCCGTAACACTGCAGCAGGTGC TGCCCGCGCTTGCACCGTCCGAAGAAAAGCGCGGCCTAAA GCGCGAGTCTGGTGACTTGGCACCCACCGTGCAGCTGATG GTACCCAAGCGCCAGCGACTGGAAGATGTCTTGGAAAAAA TGACCGTGGAACCTGGGCTGGAGCCCGAGGTCCGCGTGCG GCCAATCAAGCAGGTGGCGCCGGGACTGGGCGTGCAGACC GTGGACGTTCAGATACCCACTACCAGTAGCACCAGTATTG CCACCGCCACAGAGGGCATGGAGACACAAACGTCCCCGGT TGCCTCAGCGGTGGCGGATGCCGCGGTGCAGGCGGTCGCT GCGGCCGCGTCCAAGACCTCTACGGAGGTGCAAACGGACC CGTGGATGTTTCGCGTTTCAGCCCCCCGGCGCCCGCGCCGT TCGAGGAAGTACGGCGCCGCCAGCGCGCTACTGCCCGAAT ATGCCCTACATCCTTCCATTGCGCCTACCCCCGGCTATCGT GGCTACACCTACCGCCCCAGAAGACGAGCAACTACCCGAC GCCGAACCACCACTGGAACCCGCCGCCGCCGTCGCCGTCG CCAGCCCGTGCTGGCCCCGATTTCCGTGCGCAGGGTGGCTC GCGAAGGAGGCAGGACCCTGGTGCTGCCAACAGCGCGCTA CCACCCCAGCATCGTTTAAAAGCCGGTCTTTGTGGTTCTTG CAGATATGGCCCTCACCTGCCGCCTCCGTTTCCCGGTGCCG GGATTCCGAGGAAGAATGCACCGTAGGAGGGGCATGGCCG GCCACGGCCTGACGGGCGGCATGCGTCGTGCGCACCACCG GCGGCGGCGCGCGTCGCACCGTCGCATGCGCGGCGGTATC CTGCCCCTCCTTATTCCACTGATCGCCGCGGCGATTGGCGC CGTGCCCGGAATTGCATCCGTGGCCTTGCAGGCGCAGAGA CACTGATTAAAAACAAGTTGCATGTGGAAAAATCAAAATA AAAAGTCTGGACTCTCACGCTCGCTTGGTCCTGTAACTATT TTGTAGAATGGAAGACATCAACTTTGCGTCTCTGGCCCCGC GACACGGCTCGCGCCCGTTCATGGGAAACTGGCAAGATAT CGGCACCAGCAATATGAGCGGTGGCGCCTTCAGCTGGGGC TCGCTGTGGAGCGGCATTAAAAATTTCGGTTCCACCGTTAA GAACTATGGCAGCAAGGCCTGGAACAGCAGCACAGGCCA GATGCTGAGGGATAAGTTGAAAGAGCAAAATTTCCAACAA AAGGTGGTAGATGGCCTGGCCTCTGGCATTAGCGGGGTGG TGGACCTGGCCAACCAGGCAGTGCAAAATAAGATTAACAG TAAGCTTGATCCCCGCCCTCCCGTAGAGGAGCCTCCACCGG CCGTGGAGACAGTGTCTCCAGAGGGGCGTGGCGAAAAGCG TCCGCGCCCCGACAGGGAAGAAACTCTGGTGACGCAAATA GACGAGCCTCCCTCGTACGAGGAGGCACTAAAGCAAGGCC TGCCCACCACCCGTCCCATCGCGCCCATGGCTACCGGAGTG CTGGGCCAGCACACACCCGTAACGCTGGACCTGCCTCCCC CCGCCGACACCCAGCAGAAACCTGTGCTGCCAGGCCCGAC CGCCGTTGTTGTAACCCGTCCTAGCCGCGCGTCCCTGCGCC GCGCCGCCAGCGGTCCGCGATCGTTGCGGCCCGTAGCCAG TGGCAACTGGCAAAGCACACTGAACAGCATCGTGGGTCTG GGGGTGCAATCCCTGAAGCGCCGACGATGCTTCTGATAGC TAACGTGTCGTATGTGTGTCATGTATGCGTCCATGTCGCCG CCAGAGGAGCTGCTGAGCCGCCGCGCGCCCGCTTTCCAAG ATGGCTACCCCTTCGATGATGCCGCAGTGGTCTTACATGCA CATCTCGGGCCAGGACGCCTCGGAGTACCTGAGCCCCGGG CTGGTGCAGTTTGCCCGCGCCACCGAGACGTACTTCAGCCT GAATAACAAGTTTAGAAACCCCACGGTGGCGCCTACGCAC GACGTGACCACAGACCGGTCCCAGCGTTTGACGCTGCGGT TCATCCCTGTGGACCGTGAGGATACTGCGTACTCGTACAAG GCGCGGTTCACCCTAGCTGTGGGTGATAACCGTGTGCTGG ACATGGCTTCCACGTACTTTGACATCCGCGGCGTGCTGGAC AGGGGCCCTACTTTTAAGCCCTACTCTGGCACTGCCTACAA CGCCCTGGCTCCCAAGGGTGCCCCAAATCCTTGCGAATGG GATGAAGCTGCTACTGCTCTTGAAATAAACCTAGAAGAAG AGGACGATGACAACGAAGACGAAGTAGACGAGCAAGCTG AGCAGCAAAAAACTCACGTATTTGGGCAGGCGCCTTATTC TGGTATAAATATTACAAAGGAGGGTATTCAAATAGGTGTC GAAGGTCAAACACCTAAATATGCCGATAAAACATTTCAAC CTGAACCTCAAATAGGAGAATCTCAGTGGTACGAAACAGA AATTAATCATGCAGCTGGGAGAGTCCTAAAAAAGACTACC CCAATGAAACCATGTTACGGTTCATATGCAAAACCCACAA ATGAAAATGGAGGGCAAGGCATTCTTGTAAAGCAACAAAA TGGAAAGCTAGAAAGTCAAGTGGAAATGCAATTTTTCTCA ACTACTGAGGCAGCCGCAGGCAATGGTGATAACTTGACTC CTAAAGTGGTATTGTACAGTGAAGATGTAGATATAGAAAC CCCAGACACTCATATTTCTTACATGCCCACTATTAAGGAAG GTAACTCACGAGAACTAATGGGCCAACAATCTATGCCCAA CAGGCCTAATTACATTGCTTTTAGGGACAATTTTATTGGTC TAATGTATTACAACAGCACGGGTAATATGGGTGTTCTGGC GGGCCAAGCATCGCAGTTGAATGCTGTTGTAGATTTGCAA GACAGAAACACAGAGCTTTCATACCAGCTTTTGCTTGATTC CATTGGTGATAGAACCAGGTACTTTTCTATGTGGAATCAGG CTGTTGACAGCTATGATCCAGATGTTAGAATTATTGAAAAT CATGGAACTGAAGATGAACTTCCAAATTACTGCTTTCCACT GGGAGGTGTGATTAATACAGAGACTCTTACCAAGGTAAAA CCTAAAACAGGTCAGGAAAATGGATGGGAAAAAGATGCT ACAGAATTTTCAGATAAAAATGAAATAAGAGTTGGAAATA ATTTTGCCATGGAAATCAATCTAAATGCCAACCTGTGGAG AAATTTCCTGTACTCCAACATAGCGCTGTATTTGCCCGACA AGCTAAAGTACAGTCCTTCCAACGTAAAAATTTCTGATAAC CCAAACACCTACGACTACATGAACAAGCGAGTGGTGGCTC CCGGGCTAGTGGACTGCTACATTAACCTTGGAGCACGCTG GTCCCTTGACTATATGGACAACGTCAACCCATTTAACCACC ACCGCAATGCTGGCCTGCGCTACCGCTCAATGTTGCTGGGC AATGGTCGCTATGTGCCCTTCCACATCCAGGTGCCTCAGAA GTTCTTTGCCATTAAAAACCTCCTTCTCCTGCCGGGCTCAT ACACCTACGAGTGGAACTTCAGGAAGGATGTTAACATGGT TCTGCAGAGCTCCCTAGGAAATGACCTAAGGGTTGACGGA GCCAGCATTAAGTTTGATAGCATTTGCCTTTACGCCACCTT CTTCCCCATGGCCCACAACACCGCCTCCACGCTTGAGGCCA TGCTTAGAAACGACACCAACGACCAGTCCTTTAACGACTA TCTCTCCGCCGCCAACATGCTCTACCCTATACCCGCCAACG CTACCAACGTGCCCATATCCATCCCCTCCCGCAACTGGGCG GCTTTCCGCGGCTGGGCCTTCACGCGCCTTAAGACTAAGGA AACCCCATCACTGGGCTCGGGCTACGACCCTTATTACACCT ACTCTGGCTCTATACCCTACCTAGATGGAACCTTTTACCTC AACCACACCTTTAAGAAGGTGGCCATTACCTTTGACTCTTC TGTCAGCTGGCCTGGCAATGACCGCCTGCTTACCCCCAACG AGTTTGAAATTAAGCGCTCAGTTGACGGGGAGGGTTACAA CGTTGCCCAGTGTAACATGACCAAAGACTGGTTCCTGGTAC AAATGCTAGCTAACTATAACATTGGCTACCAGGGCTTCTAT ATCCCAGAGAGCTACAAGGACCGCATGTACTCCTTCTTTAG AAACTTCCAGCCCATGAGCCGTCAGGTGGTGGATGATACT AAATACAAGGACTACCAACAGGTGGGCATCCTACACCAAC ACAACAACTCTGGATTTGTTGGCTACCTTGCCCCCACCATG CGCGAAGGACAGGCCTACCCTGCTAACTTCCCCTATCCGCT TATAGGCAAGACCGCAGTTGACAGCATTACCCAGAAAAAG TTTCTTTGCGATCGCACCCTTTGGCGCATCCCATTCTCCAGT AACTTTATGTCCATGGGCGCACTCACAGACCTGGGCCAAA ACCTTCTCTACGCCAACTCCGCCCACGCGCTAGACATGACT TTTGAGGTGGATCCCATGGACGAGCCCACCCTTCTTTATGT TTTGTTTGAAGTCTTTGACGTGGTCCGTGTGCACCAGCCGC ACCGCGGCGTCATCGAAACCGTGTACCTGCGCACGCCCTTC TCGGCCGGCAACGCCACAACATAAAGAAGCAAGCAACATC AACAACAGCTGCCGCCATGGGCTCCAGTGAGCAGGAACTG AAAGCCATTGTCAAAGATCTTGGTTGTGGGCCATATTTTTT GGGCACCTATGACAAGCGCTTTCCAGGCTTTGTTTCTCCAC ACAAGCTCGCCTGCGCCATAGTCAATACGGCCGGTCGCGA GACTGGGGGCGTACACTGGATGGCCTTTGCCTGGAACCCG CACTCAAAAACATGCTACCTCTTTGAGCCCTTTGGCTTTTC TGACCAGCGACTCAAGCAGGTTTACCAGTTTGAGTACGAG TCACTCCTGCGCCGTAGCGCCATTGCTTCTTCCCCCGACCG CTGTATAACGCTGGAAAAGTCCACCCAAAGCGTACAGGGG CCCAACTCGGCCGCCTGTGGACTATTCTGCTGCATGTTTCT CCACGCCTTTGCCAACTGGCCCCAAACTCCCATGGATCACA ACCCCACCATGAACCTTATTACCGGGGTACCCAACTCCATG CTCAACAGTCCCCAGGTACAGCCCACCCTGCGTCGCAACC AGGAACAGCTCTACAGCTTCCTGGAGCGCCACTCGCCCTA CTTCCGCAGCCACAGTGCGCAGATTAGGAGCGCCACTTCTT TTTGTCACTTGAAAAACATGTAAAAATAATGTACTAGAGA CACTTTCAATAAAGGCAAATGCTTTTATTTGTACACTCTCG GGTGATTATTTACCCCCACCCTTGCCGTCTGCGCCGTTTAA AAATCAAAGGGGTTCTGCCGCGCATCGCTATGCGCCACTG GCAGGGACACGTTGCGATACTGGTGTTTAGTGCTCCACTTA AACTCAGGCACAACCATCCGCGGCAGCTCGGTGAAGTTTT CACTCCACAGGCTGCGCACCATCACCAACGCGTTTAGCAG GTCGGGCGCCGATATCTTGAAGTCGCAGTTGGGGCCTCCG CCCTGCGCGCGCGAGTTGCGATACACAGGGTTGCAGCACT GGAACACTATCAGCGCCGGGTGGTGCACGCTGGCCAGCAC GCTCTTGTCGGAGATCAGATCCGCGTCCAGGTCCTCCGCGT TGCTCAGGGCGAACGGAGTCAACTTTGGTAGCTGCCTTCCC AAAAAGGGCGCGTGCCCAGGCTTTGAGTTGCACTCGCACC GTAGTGGCATCAAAAGGTGACCGTGCCCGGTCTGGGCGTT AGGATACAGCGCCTGCATAAAAGCCTTGATCTGCTTAAAA GCCACCTGAGCCTTTGCGCCTTCAGAGAAGAACATGCCGC AAGACTTGCCGGAAAACTGATTGGCCGGACAGGCCGCGTC GTGCACGCAGCACCTTGCGTCGGTGTTGGAGATCTGCACC ACATTTCGGCCCCACCGGTTCTTCACGATCTTGGCCTTGCT AGACTGCTCCTTCAGCGCGCGCTGCCCGTTTTCGCTCGTCA CATCCATTTCAATCACGTGCTCCTTATTTATCATAATGCTTC CGTGTAGACACTTAAGCTCGCCTTCGATCTCAGCGCAGCGG TGCAGCCACAACGCGCAGCCCGTGGGCTCGTGATGCTTGT AGGTCACCTCTGCAAACGACTGCAGGTACGCCTGCAGGAA TCGCCCCATCATCGTCACAAAGGTCTTGTTGCTGGTGAAGG TCAGCTGCAACCCGCGGTGCTCCTCGTTCAGCCAGGTCTTG CATACGGCCGCCAGAGCTTCCACTTGGTCAGGCAGTAGTTT GAAGTTCGCCTTTAGATCGTTATCCACGTGGTACTTGTCCA TCAGCGCGCGCGCAGCCTCCATGCCCTTCTCCCACGCAGAC ACGATCGGCACACTCAGCGGGTTCATCACCGTAATTTCACT TTCCGCTTCGCTGGGCTCTTCCTCTTCCTCTTGCGTCCGCAT ACCACGCGCCACTGGGTCGTCTTCATTCAGCCGCCGCACTG TGCGCTTACCTCCTTTGCCATGCTTGATTAGCACCGGTGGG TTGCTGAAACCCACCATTTGTAGCGCCACATCTTCTCTTTCT TCCTCGCTGTCCACGATTACCTCTGGTGATGGCGGGCGCTC GGGCTTGGGAGAAGGGCGCTTCTTTTTCTTCTTGGGCGCAA TGGCCAAATCCGCCGCCGAGGTCGATGGCCGCGGGCTGGG TGTGCGCGGCACCAGCGCGTCTTGTGATGAGTCTTCCTCGT CCTCGGACTCGATACGCCGCCTCATCCGCTTTTTTGGGGGC GCCCGGGGAGGCGGCGGCGACGGGGACGGGGACGACACG TCCTCCATGGTTGGGGGACGTCGCGCCGCACCGCGTCCGC GCTCGGGGGTGGTTTCGCGCTGCTCCTCTTCCCGACTGGCC ATTTCCTTCTCCTATAGGCAGAAAAAGATCATGGAGTCAGT CGAGAAGAAGGACAGCCTAACCGCCCCCTCTGAGTTCGCC ACCACCGCCTCCACCGATGCCGCCAACGCGCCTACCACCTT CCCCGTCGAGGCACCCCCGCTTGAGGAGGAGGAAGTGATT ATCGAGCAGGACCCAGGTTTTGTAAGCGAAGACGACGAGG ACCGCTCAGTACCAACAGAGGATAAAAAGCAAGACCAGG ACAACGCAGAGGCAAACGAGGAACAAGTCGGGCGGGGGG ACGAAAGGCATGGCGACTACCTAGATGTGGGAGACGACGT GCTGTTGAAGCATCTGCAGCGCCAGTGCGCCATTATCTGCG ACGCGTTGCAAGAGCGCAGCGATGTGCCCCTCGCCATAGC GGATGTCAGCCTTGCCTACGAACGCCACCTATTCTCACCGC GCGTACCCCCCAAACGCCAAGAAAACGGCACATGCGAGCC CAACCCGCGCCTCAACTTCTACCCCGTATTTGCCGTGCCAG AGGTGCTTGCCACCTATCACATCTTTTTCCAAAACTGCAAG ATACCCCTATCCTGCCGTGCCAACCGCAGCCGAGCGGACA AGCAGCTGGCCTTGCGGCAGGGCGCTGTCATACCTGATAT CGCCTCGCTCAACGAAGTGCCAAAAATCTTTGAGGGTCTTG GACGCGACGAGAAGCGCGCGGCAAACGCTCTGCAACAGG AAAACAGCGAAAATGAAAGTCACTCTGGAGTGTTGGTGGA ACTCGAGGGTGACAACGCGCGCCTAGCCGTACTAAAACGC AGCATCGAGGTCACCCACTTTGCCTACCCGGCACTTAACCT ACCCCCCAAGGTCATGAGCACAGTCATGAGTGAGCTGATC GTGCGCCGTGCGCAGCCCCTGGAGAGGGATGCAAATTTGC AAGAACAAACAGAGGAGGGCCTACCCGCAGTTGGCGACG AGCAGCTAGCGCGCTGGCTTCAAACGCGCGAGCCTGCCGA CTTGGAGGAGCGACGCAAACTAATGATGGCCGCAGTGCTC GTTACCGTGGAGCTTGAGTGCATGCAGCGGTTCTTTGCTGA CCCGGAGATGCAGCGCAAGCTAGAGGAAACATTGCACTAC ACCTTTCGACAGGGCTACGTACGCCAGGCCTGCAAGATCT CCAACGTGGAGCTCTGCAACCTGGTCTCCTACCTTGGAATT TTGCACGAAAACCGCCTTGGGCAAAACGTGCTTCATTCCAC GCTCAAGGGCGAGGCGCGCCGCGACTACGTCCGCGACTGC GTTTACTTATTTCTATGCTACACCTGGCAGACGGCCATGGG CGTTTGGCAGCAGTGCTTGGAGGAGTGCAACCTCAAGGAG CTGCAGAAACTGCTAAAGCAAAACTTGAAGGACCTATGGA CGGCCTTCAACGAGCGCTCCGTGGCCGCGCACCTGGCGGA CATCATTTTCCCCGAACGCCTGCTTAAAACCCTGCAACAGG GTCTGCCAGACTTCACCAGTCAAAGCATGTTGCAGAACTTT AGGAACTTTATCCTAGAGCGCTCAGGAATCTTGCCCGCCAC CTGCTGTGCACTTCCTAGCGACTTTGTGCCCATTAAGTACC GCGAATGCCCTCCGCCGCTTTGGGGCCACTGCTACCTTCTG CAGCTAGCCAACTACCTTGCCTACCACTCTGACATAATGGA AGACGTGAGCGGTGACGGTCTACTGGAGTGTCACTGTCGC TGCAACCTATGCACCCCGCACCGCTCCCTGGTTTGCAATTC GCAGCTGCTTAACGAAAGTCAAATTATCGGTACCTTTGAGC TGCAGGGTCCCTCGCCTGACGAAAAGTCCGCGGCTCCGGG GTTGAAACTCACTCCGGGGCTGTGGACGTCGGCTTACCTTC GCAAATTTGTACCTGAGGACTACCACGCCCACGAGATTAG GTTCTACGAAGACCAATCCCGCCCGCCTAATGCGGAGCTT ACCGCCTGCGTCATTACCCAGGGCCACATTCTTGGCCAATT GCAAGCCATCAACAAAGCCCGCCAAGAGTTTCTGCTACGA AAGGGACGGGGGGTTTACTTGGACCCCCAGTCCGGCGAGG AGCTCAACCCAATCCCCCCGCCGCCGCAGCCCTATCAGCA GCAGCCGCGGGCCCTTGCTTCCCAGGATGGCACCCAAAAA GAAGCTGCAGCTGCCGCCGCCACCCACGGACGAGGAGGAA TACTGGGACAGTCAGGCAGAGGAGGTTTTGGACGAGGAGG AGGAGGACATGATGGAAGACTGGGAGAGCCTAGACGAGG AAGCTTCCGAGGTCGAAGAGGTGTCAGACGAAACACCGTC ACCCTCGGTCGCATTCCCCTCGCCGGCGCCCCAGAAATCGG CAACCGGTTCCAGCATGGCTACAACCTCCGCTCCTCAGGCG CCGCCGGCACTGCCCGTTCGCCGACCCAACCGTAGATGGG ACACCACTGGAACCAGGGCCGGTAAGTCCAAGCAGCCGCC GCCGTTAGCCCAAGAGCAACAACAGCGCCAAGGCTACCGC TCATGGCGCGGGCACAAGAACGCCATAGTTGCTTGCTTGC AAGACTGTGGGGGCAACATCTCCTTCGCCCGCCGCTTTCTT CTCTACCATCACGGCGTGGCCTTCCCCCGTAACATCCTGCA TTACTACCGTCATCTCTACAGCCCATACTGCACCGGCGGCA GCGGCAGCAACAGCAGCGGCCACACAGAAGCAAAGGCGA CCGGATAGCAAGACTCTGACAAAGCCCAAGAAATCCACAG CGGCGGCAGCAGCAGGAGGAGGAGCGCTGCGTCTGGCGCC CAACGAACCCGTATCGACCCGCGAGCTTAGAAACAGGATT TTTCCCACTCTGTATGCTATATTTCAACAGAGCAGGGGCCA AGAACAAGAGCTGAAAATAAAAAACAGGTCTCTGCGATCC CTCACCCGCAGCTGCCTGTATCACAAAAGCGAAGATCAGC TTCGGCGCACGCTGGAAGACGCGGAGGCTCTCTTCAGTAA ATACTGCGCGCTGACTCTTAAGGACTAGTTTCGCGCCCTTT CTCAAATTTAAGCGCGAAAACTACGTCATCTCCAGCGGCC ACACCCGGCGCCAGCACCTGTTGTCAGCGCCATTATGAGC AAGGAAATTCCCACGCCCTACATGTGGAGTTACCAGCCAC AAATGGGACTTGCGGCTGGAGCTGCCCAAGACTACTCAAC CCGAATAAACTACATGAGCGCGGGACCCCACATGATATCC CGGGTCAACGGAATACGCGCCCACCGAAACCGAATTCTCC TGGAACAGGCGGCTATTACCACCACACCTCGTAATAACCTT AATCCCCGTAGTTGGCCCGCTGCCCTGGTGTACCAGGAAA GTCCCGCTCCCACCACTGTGGTACTTCCCAGAGACGCCCAG GCCGAAGTTCAGATGACTAACTCAGGGGCGCAGCTTGCGG GCGGCTTTCGTCACAGGGTGCGGTCGCCCGGGCAGGGTAT AACTCACCTGACAATCAGAGGGCGAGGTATTCAGCTCAAC GACGAGTCGGTGAGCTCCTCGCTTGGTCTCCGTCCGGACGG GACATTTCAGATCGGCGGCGCCGGCCGCTCTTCATTCACGC CTCGTCAGGCAATCCTAACTCTGCAGACCTCGTCCTCTGAG CCGCGCTCTGGAGGCATTGGAACTCTGCAATTTATTGAGGA GTTTGTGCCATCGGTCTACTTTAACCCCTTCTCGGGACCTC CCGGCCACTATCCGGATCAATTTATTCCTAACTTTGACGCG GTAAAGGACTCGGCGGACGGCTACGACTGAATGTTAAGTG GAGAGGCAGAGCAACTGCGCCTGAAACACCTGGTCCACTG TCGCCGCCACAAGTGCTTTGCCCGCGACTCCGGTGAGTTTT GCTACTTTGAATTGCCCGAGGATCATATCGAGGGCCCGGC GCACGGCGTCCGGCTTACCGCCCAGGGAGAGCTTGCCCGT AGCCTGATTCGGGAGTTTACCCAGCGCCCCCTGCTAGTTGA GCGGGACAGGGGACCCTGTGTTCTCACTGTGATTTGCAACT GTCCTAACCCTGGATTACATCAAGATCCTCTAGTTAATGTC AGGTCGCCTAAGTCGATTAACTAGAGTACCCGGGGATCTT ATTCCCTTTAACTAATAAAAAAAAATAATAAAGCATCACTT ACTTAAAATCAGTTAGCAAATTTCTGTCCAGTTTATTCAGC AGCACCTCCTTGCCCTCCTCCCAGCTCTGGTATTGCAGCTT CCTCCTGGCTGCAAACTTTCTCCACAATCTAAATGGAATGT CAGTTTCCTCCTGTTCCTGTCCATCCGCACCCACTATCTTCA TGTTGTTGCAGATGAAGCGCGCAAGACCGTCTGAAGATAC CTTCAACCCCGTGTATCCATATGACACGGAAACCGGTCCTC CAACTGTGCCTTTTCTTACTCCTCCCTTTGTATCCCCCAATG GGTTTCAAGAGAGTCCCCCTGGGGTACTCTCTTTGCGCCTA TCCGAACCTCTAGTTACCTCCAATGGCATGCTTGCGCTCAA AATGGGCAACGGCCTCTCTCTGGACGAGGCCGGCAACCTT ACCTCCCAAAATGTAACCACTGTGAGCCCACCTCTCAAAA AAACCAAGTCAAACATAAACCTGGAAATATCTGCACCCCT CACAGTTACCTCAGAAGCCCTAACTGTGGCTGCCGCCGCA CCTCTAATGGTCGCGGGCAACACACTCACCATGCAATCAC AGGCCCCGCTAACCGTGCACGACTCCAAACTTAGCATTGC CACCCAAGGACCCCTCACAGTGTCAGAAGGAAAGCTAGCC CTGCAAACATCAGGCCCCCTCACCACCACCGATAGCAGTA CCCTTACTATCACTGCCTCACCCCCTCTAACTACTGCCACT GGTAGCTTGGGCATTGACTTGAAAGAGCCCATTTATACAC AAAATGGAAAACTAGGACTAAAGTACGGGGCTCCTTTGCA TGTAACAGACGACCTAAACACTTTGACCGTAGCAACTGGT CCAGGTGTGACTATTAATAATACTTCCTTGCAAACTAAAGT TACTGGAGCCTTGGGTTTTGATTCACAAGGCAATATGCAAC TTAATGTAGCAGGAGGACTAAGGATTGATTCTCAAAACAG ACGCCTTATACTTGATGTTAGTTATCCGTTTGATGCTCAAA ACCAACTAAATCTAAGACTAGGACAGGGCCCTCTTTTTATA AACTCAGCCCACAACTTGGATATTAACTACAACAAAGGCC TTTACTTGTTTACAGCTTCAAACAATTCCAAAAAGCTTGAG GTTAACCTAAGCACTGCCAAGGGGTTGATGTTTGACGCTAC AGCCATAGCCATTAATGCAGGAGATGGGCTTGAATTTGGT TCACCTAATGCACCAAACACAAATCCCCTCAAAACAAAAA TTGGCCATGGCCTAGAATTTGATTCAAACAAGGCTATGGTT CCTAAACTAGGAACTGGCCTTAGTTTTGACAGCACAGGTG CCATTACAGTAGGAAACAAAAATAATGATAAGCTAACTTT GTGGACCACACCAGCTCCATCTCCTAACTGTAGACTAAATG CAGAGAAAGATGCTAAACTCACTTTGGTCTTAACAAAATG TGGCAGTCAAATACTTGCTACAGTTTCAGTTTTGGCTGTTA AAGGCAGTTTGGCTCCAATATCTGGAACAGTTCAAAGTGC TCATCTTATTATAAGATTTGACGAAAATGGAGTGCTACTAA ACAATTCCTTCCTGGACCCAGAATATTGGAACTTTAGAAAT GGAGATCTTACTGAAGGCACAGCCTATACAAACGCTGTTG GATTTATGCCTAACCTATCAGCTTATCCAAAATCTCACGGT AAAACTGCCAAAAGTAACATTGTCAGTCAAGTTTACTTAA ACGGAGACAAAACTAAACCTGTAACACTAACCATTACACT AAACGGTACACAGGAAACAGGAGACACAACTCCAAGTGC ATACTCTATGTCATTTTCATGGGACTGGTCTGGCCACAACT ACATTAATGAAATATTTGCCACATCCTCTTACACTTTTTCAT ACATTGCCCAAGAATAAAGAATCGTTTGTGTTATGTTTCAA CGTGTTTATTTTTCAATTGCAGAAAATTTCAAGTCATTTTTC ATTCAGTAGTATAGCCCCACCACCACATAGCTTATACAGAT CACCGTACCTTAATCAAACTCACAGAACCCTAGTATTCAAC CTGCCACCTCCCTCCCAACACACAGAGTACACAGTCCTTTC TCCCCGGCTGGCCTTAAAAAGCATCATATCATGGGTAACA GACATATTCTTAGGTGTTATATTCCACACGGTTTCCTGTCG AGCCAAACGCTCATCAGTGATATTAATAAACTCCCCGGGC AGCTCACTTAAGTTCATGTCGCTGTCCAGCTGCTGAGCCAC AGGCTGCTGTCCAACTTGCGGTTGCTTAACGGGCGGCGAA GGAGAAGTCCACGCCTACATGGGGGTAGAGTCATAATCGT GCATCAGGATAGGGCGGTGGTGCTGCAGCAGCGCGCGAAT AAACTGCTGCCGCCGCCGCTCCGTCCTGCAGGAATACAAC ATGGCAGTGGTCTCCTCAGCGATGATTCGCACCGCCCGCA GCATAAGGCGCCTTGTCCTCCGGGCACAGCAGCGCACCCT GATCTCACTTAAATCAGCACAGTAACTGCAGCACAGCACC ACAATATTGTTCAAAATCCCACAGTGCAAGGCGCTGTATCC AAAGCTCATGGCGGGGACCACAGAACCCACGTGGCCATCA TACCACAAGCGCAGGTAGATTAAGTGGCGACCCCTCATAA ACACGCTGGACATAAACATTACCTCTTTTGGCATGTTGTAA TTCACCACCTCCCGGTACCATATAAACCTCTGATTAAACAT GGCGCCATCCACCACCATCCTAAACCAGCTGGCCAAAACC TGCCCGCCGGCTATACACTGCAGGGAACCGGGACTGGAAC AATGACAGTGGAGAGCCCAGGACTCGTAACCATGGATCAT CATGCTCGTCATGATATCAATGTTGGCACAACACAGGCAC ACGTGCATACACTTCCTCAGGATTACAAGCTCCTCCCGCGT TAGAACCATATCCCAGGGAACAACCCATTCCTGAATCAGC GTAAATCCCACACTGCAGGGAAGACCTCGCACGTAACTCA CGTTGTGCATTGTCAAAGTGTTACATTCGGGCAGCAGCGG ATGATCCTCCAGTATGGTAGCGCGGGTTTCTGTCTCAAAAG GAGGTAGACGATCCCTACTGTACGGAGTGCGCCGAGACAA CCGAGATCGTGTTGGTCGTAGTGTCATGCCAAATGGAACG CCGGACGTAGTCATATTTCCTGAAGCAAAACCAGGTGCGG GCGTGACAAACAGATCTGCGTCTCCGGTCTCGCCGCTTAGA TCGCTCTGTGTAGTAGTTGTAGTATATCCACTCTCTCAAAG CATCCAGGCGCCCCCTGGCTTCGGGTTCTATGTAAACTCCT TCATGCGCCGCTGCCCTGATAACATCCACCACCGCAGAAT AAGCCACACCCAGCCAACCTACACATTCGTTCTGCGAGTC ACACACGGGAGGAGCGGGAAGAGCTGGAAGAACCATGTT TTTTTTTTTATTCCAAAAGATTATCCAAAACCTCAAAATGA AGATCTATTAAGTGAACGCGCTCCCCTCCGGTGGCGTGGTC AAACTCTACAGCCAAAGAACAGATAATGGCATTTGTAAGA TGTTGCACAATGGCTTCCAAAAGGCAAACGGCCCTCACGT CCAAGTGGACGTAAAGGCTAAACCCTTCAGGGTGAATCTC CTCTATAAACATTCCAGCACCTTCAACCATGCCCAAATAAT TCTCATCTCGCCACCTTCTCAATATATCTCTAAGCAAATCC CGAATATTAAGTCCGGCCATTGTAAAAATCTGCTCCAGAG CGCCCTCCACCTTCAGCCTCAAGCAGCGAATCATGATTGCA AAAATTCAGGTTCCTCACAGACCTGTATAAGATTCAAAAG CGGAACATTAACAAAAATACCGCGATCCCGTAGGTCCCTT CGCAGGGCCAGCTGAACATAATCGTGCAGGTCTGCACGGA CCAGCGCGGCCACTTCCCCGCCAGGAACCATGACAAAAGA ACCCACACTGATTATGACACGCATACTCGGAGCTATGCTA ACCAGCGTAGCCCCGATGTAAGCTTGTTGCATGGGCGGCG ATATAAAATGCAAGGTGCTGCTCAAAAAATCAGGCAAAGC CTCGCGCAAAAAAGAAAGCACATCGTAGTCATGCTCATGC AGATAAAGGCAGGTAAGCTCCGGAACCACCACAGAAAAA GACACCATTTTTCTCTCAAACATGTCTGCGGGTTTCTGCAT AAACACAAAATAAAATAACAAAAAAACATTTAAACATTAG AAGCCTGTCTTACAACAGGAAAAACAACCCTTATAAGCAT AAGACGGACTACGGCCATGCCGGCGTGACCGTAAAAAAAC TGGTCACCGTGATTAAAAAGCACCACCGACAGCTCCTCGG TCATGTCCGGAGTCATAATGTAAGACTCGGTAAACACATC AGGTTGATTCACATCGGTCAGTGCTAAAAAGCGACCGAAA TAGCCCGGGGGAATACATACCCGCAGGCGTAGAGACAACA TTACAGCCCCCATAGGAGGTATAACAAAATTAATAGGAGA GAAAAACACATAAACACCTGAAAAACCCTCCTGCCTAGGC AAAATAGCACCCTCCCGCTCCAGAACAACATACAGCGCTT CCACAGCGGCAGCCATAACAGTCAGCCTTACCAGTAAAAA AGAAAACCTATTAAAAAAACACCACTCGACACGGCACCAG CTCAATCAGTCACAGTGTAAAAAAGGGCCAAGTGCAGAGC GAGTATATATAGGACTAAAAAATGACGTAACGGTTAAAGT CCACAAAAAACACCCAGAAAACCGCACGCGAACCTACGCC CAGAAACGAAAGCCAAAAAACCCACAACTTCCTCAAATCG TCACTTCCGTTTTCCCACGTTACGTCACTTCCCATTTTAAGA AAACTACAATTCCCAACACATACAAGTTACTCCGCCCTAA AACCTACGTCACCCGCCCCGTTCCCACGCCCCGCGCCACGT CACAAACTCCACCCCCTCATTATCATATTGGCTTCAATCCA AAATAAGGTATATTATTGATGATG 95 Full nucleotide (human IL- TTTTGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTT sequence of 12 insert TGTGACGTGGCGCGGGGCGTGGGAACGGGGCGGGTGACGT TRZ627 hIL- sequence in AGTAGTGTGGCGGAAGTGTGATGTTGCAAGTGTGGCGGAA 12 virus CACATGTAAGCGACGGATGTGGCAAAAGTGACGTTTTTGG bold codon TGTGCGCCGGTGTTTTGGGCGTAACCGAGTAAGATTTGGCC (beginning at ATTTTCGCGGGAAAACTGAATAAGAGGAAGTGAAATCTGA optimized) ATAATTTTGTGTTACTCATAGCGCGTAATATTTGTCTAGGG Ad5 5′ end CCGCGGGGACTTTGACCGTTTACGTGGAGACTCGCCCAGG ITR) TGTTTTTCTCAGGTGTTTTCCGCGTTCCGGGTCAAAGTTGG CGTTTTATTATTATAGTCAGCTGACGTGTAGTGTATTTATA CCCGGTGAGTTCCTCAAGAGGCCACTCTTGAGTGCCAGCG AGTAGAGTTTTCTCCTCCGAGCCGCTCCGACACCGGGACTG AAAATGAGACATATTATCTGCCACGGAGGTGTTATTACCG AAGAAATGGCCGCCAGTCTTTTGGACCAGCTGATCGAAGA GGTACTGGCTGATAATCTTCCACCTCCTAGCCATTTTGAAC CACCTACCCTTCACGAACTGTATGATTTAGACGTGACGGCC CCCGAAGATCCCAACGAGGAGGCGGTTTCGCAGATTTTTC CCGACTCTGTAATGTTGGCGGTGCAGGAAGGGATTGACTT ACTCACTTTTCCGCCGGCGCCCGGTTCTCCGGAGCCGCCTC ACCTTTCCCGGCAGCCCGAGCAGCCGGAGCAGAGAGCCTT GGGTCCGGTTTCTATGCCAAACCTTGTACCGGAGGTGATCG ATCTTACCTGCCACGAGGCTGGCTTTCCACCCAGTGACGAC GAGGATGAAGAGGGTGAGGAGTTTGTGTTAGATTATGTGG AGCACCCCGGGCACGGTTGCAGGTCTTGTCATTATCACCGG AGGAATACGGGGGACCCAGATATTATGTGTTCGCTTTGCTA TATGAGGACCTGTGGCATGTTTGTCTACAGTAAGTGAAAAT TATGGGCAGTGGGTGATAGAGTGGTGGGTTTGGTGTGGTA ATTTTTTTTTTAATTTTTACAGTTTTGTGGTTTAAAGAATTT TGTATTGTGATTTTTTTAAAAGGTCCTGTGTCTGAACCTGA GCCTGAGCCCGAGCCAGAACCGGAGCCTGCAAGACCTACC CGCCGTCCTAAAATGGCGCCTGCTATCCTGAGACGCCCGA CATCACCTGTGTCTAGAGAATGCAATAGTAGTACGGATAG CTGTGACTCCGGTCCTTCTAACACACCTCCTGAGATACACC CGGTGGTCCCGCTGTGCCCCATTAAACCAGTTGCCGTGAGA GTTGGTGGGCGTCGCCAGGCTGTGGAATGTATCGAGGACT TGCTTAACGAGCCTGGGCAACCTTTGGACTTGAGCTGTAAA CGCCCCAGGCCATAAGGTGTAAACCTGTGATTGCGTGTGT GGTTAACGCCTTTGTTTGCTGAATGAGTTGATGTAAGTTTA ATAAAGGGTGAGATAATGTTTAACTTGCATGGCGTGTTAA ATGGGGCGGGGCTTAAAGGGTATATAATGCGCCGTGGGCT AATCTTGGTTACATCTGACCTCGTCGACGCCACCATGTGT CACCAGCAGCTCGTGATTAGCTGGTTCAGCCTGGTGTT TCTGGCTAGCCCTCTGGTGGCCATCTGGGAGCTGAAGA AGGACGTGTACGTGGTGGAGCTCGACTGGTACCCTGAC GCTCCCGGCGAGATGGTCGTGCTGACCTGCGACACCCC TGAGGAAGATGGCATCACCTGGACCCTGGATCAAAGCT CCGAAGTGCTCGGCAGCGGCAAGACACTCACCATCCAG GTGAAAGAGTTCGGAGACGCCGGCCAGTACACCTGCCA CAAAGGCGGCGAGGTGCTGTCCCATTCCCTGCTGCTGC TGCACAAGAAAGAGGATGGCATCTGGTCCACCGACATC CTGAAGGACCAGAAGGAACCCAAGAACAAGACCTTTCT GAGATGTGAGGCCAAGAACTACAGCGGCAGGTTCACCT GCTGGTGGCTGACAACAATCTCCACCGACCTGACCTTC AGCGTCAAGAGCAGCAGGGGCAGCAGCGACCCTCAAG GCGTGACATGTGGAGCCGCTACCCTGAGCGCTGAGAGA GTCAGGGGCGACAATAAGGAGTACGAGTACTCCGTGGA ATGCCAGGAGGACTCCGCCTGCCCTGCCGCCGAAGAGT CCCTCCCTATCGAAGTGATGGTTGATGCCGTGCACAAG CTCAAGTATGAGAATTACACCAGCAGCTTTTTCATCAG GGACATCATCAAGCCCGACCCCCCCAAAAACCTCCAGC TGAAACCCCTCAAGAATAGCAGGCAGGTGGAGGTCTCC TGGGAGTATCCTGACACCTGGAGCACCCCCCACAGCTA CTTCTCCCTGACCTTCTGTGTGCAGGTGCAGGGCAAGA GCAAAAGGGAGAAGAAGGATAGGGTCTTTACCGACAAG ACCAGCGCCACAGTGATCTGCAGGAAGAACGCCAGCAT TTCCGTCAGGGCCCAGGACAGGTACTACAGCAGCAGCT GGTCCGAGTGGGCTAGCGTGCCTTGTTCCGGCGGCGG AGGATCTGGCGGAGGCGGAAGTGGCGGAGGGGGCTCT AGAAACCTCCCCGTGGCCACACCCGACCCTGGCATGTT CCCCTGCCTCCACCACAGCCAGAACCTGCTGAGAGCCG TGAGCAATATGCTGCAGAAGGCCAGGCAAACCCTGGAG TTCTACCCCTGTACCTCCGAGGAGATTGACCATGAGGA CATCACAAAGGACAAAACCAGCACCGTGGAGGCCTGTC TCCCCCTCGAACTGACCAAGAACGAGTCCTGCCTGAAC TCCAGGGAGACATCCTTCATCACCAACGGCTCCTGCCT GGCCTCCAGAAAGACCAGCTTCATGATGGCCCTCTGCC TGAGCAGCATCTACGAGGACCTCAAGATGTACCAGGTG GAGTTTAAAACAATGAACGCCAAGCTCCTCATGGACCC TAAGAGGCAGATTTTCCTCGACCAGAATATGCTGGCTG TCATTGACGAGCTGATGCAGGCCCTCAATTTCAACTCC GAGACCGTCCCCCAGAAGTCCTCCCTGGAAGAGCCCGA CTTTTACAAGACCAAGATCAAGCTCTGCATCCTGCTGC ACGCCTTCAGAATTAGAGCCGTGACCATTGACAGGGTG ATGAGCTACCTCAACGCCTCCTGATGACTCGAGTCACCA GGCGCTTTTCCAAGAGAAGGTCATCAAGACTTTGGATTTTT CCACACCGGGGCGCGCTGCGGCTGCTGTTGCTTTTTTGAGT TTTATAAAGGATAAATGGAGCGAAGAAACCCATCTGAGCG GGGGGTACCTGCTGGATTTTCTGGCCATGCATCTGTGGAGA GCGGTTGTGAGACACAAGAATCGCCTGCTACTGTTGTCTTC CGTCCGCCCGGCGATAATACCGACGGAGGAGCAGCAGCAG CAGCAGGAGGAAGCCAGGCGGCGGCGGCAGGAGCAGAGC CCATGGAACCCGAGAGCCGGCCTGGACCCTCGGGAATGAA TGTTGTACAGGTGGCTGAACTGTATCCAGAACTGAGACGC ATTTTGACAATTACAGAGGATGGGCAGGGGCTAAAGGGGG TAAAGAGGGAGCGGGGGGCTTGTGAGGCTACAGAGGAGG CTAGGAATCTAGCTTTTAGCTTAATGACCAGACACCGTCCT GAGTGTATTACTTTTCAACAGATCAAGGATAATTGCGCTAA TGAGCTTGATCTGCTGGCGCAGAAGTATTCCATAGAGCAG CTGACCACTTACTGGCTGCAGCCAGGGGATGATTTTGAGG AGGCTATTAGGGTATATGCAAAGGTGGCACTTAGGCCAGA TTGCAAGTACAAGATCAGCAAACTTGTAAATATCAGGAAT TGTTGCTACATTTCTGGGAACGGGGCCGAGGTGGAGATAG ATACGGAGGATAGGGTGGCCTTTAGATGTAGCATGATAAA TATGTGGCCGGGGGTGCTTGGCATGGACGGGGTGGTTATT ATGAATGTAAGGTTTACTGGCCCCAATTTTAGCGGTACGGT TTTCCTGGCCAATACCAACCTTATCCTACACGGTGTAAGCT TCTATGGGTTTAACAATACCTGTGTGGAAGCCTGGACCGAT GTAAGGGTTCGGGGCTGTGCCTTTTACTGCTGCTGGAAGGG GGTGGTGTGTCGCCCCAAAAGCAGGGCTTCAATTAAGAAA TGCCTCTTTGAAAGGTGTACCTTGGGTATCCTGTCTGAGGG TAACTCCAGGGTGCGCCACAATGTGGCCTCCGACTGTGGTT GCTTCATGCTAGTGAAAAGCGTGGCTGTGATTAAGCATAA CATGGTATGTGGCAACTGCGAGGACAGGGCCTCTCAGATG CTGACCTGCTCGGACGGCAACTGTCACCTGCTGAAGACCA TTCACGTAGCCAGCCACTCTCGCAAGGCCTGGCCAGTGTTT GAGCATAACATACTGACCCGCTGTTCCTTGCATTTGGGTAA CAGGAGGGGGGTGTTCCTACCTTACCAATGCAATTTGAGTC ACACTAAGATATTGCTTGAGCCCGAGAGCATGTCCAAGGT GAACCTGAACGGGGTGTTTGACATGACCATGAAGATCTGG AAGGTGCTGAGGTACGATGAGACCCGCACCAGGTGCAGAC CCTGCGAGTGTGGCGGTAAACATATTAGGAACCAGCCTGT GATGCTGGATGTGACCGAGGAGCTGAGGCCCGATCACTTG GTGCTGGCCTGCACCCGCGCTGAGTTTGGCTCTAGCGATGA AGATACAGATTGAGGTACTGAAATGTGTGGGCGTGGCTTA AGGGTGGGAAAGAATATATAAGGTGGGGGTCTTATGTAGT TTTGTATCTGTTTTGCAGCAGCCGCCGCCGCCATGAGCACC AACTCGTTTGATGGAAGCATTGTGAGCTCATATTTGACAAC GCGCATGCCCCCATGGGCCGGGGTGCGTCAGAATGTGATG GGCTCCAGCATTGATGGTCGCCCCGTCCTGCCCGCAAACTC TACTACCTTGACCTACGAGACCGTGTCTGGAACGCCGTTGG AGACTGCAGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCAC CGCCCGCGGGATTGTGACTGACTTTGCTTTCCTGAGCCCGC TTGCAAGCAGTGCAGCTTCCCGTTCATCCGCCCGCGATGAC AAGTTGACGGCTCTTTTGGCACAATTGGATTCTTTGACCCG GGAACTTAATGTCGTTTCTCAGCAGCTGTTGGATCTGCGCC AGCAGGTTTCTGCCCTGAAGGCTTCCTCCCCTCCCAATGCG GTTTAAAACATAAATAAAAAACCAGACTCTGTTTGGATTTG GATCAAGCAAGTGTCTTGCTGTCTTTATTTAGGGGTTTTGC GCGCGCGGTAGGCCCGGGACCAGCGGTCTCGGTCGTTGAG GGTCCTGTGTATTTTTTCCAGGACGTGGTAAAGGTGACTCT GGATGTTCAGATACATGGGCATAAGCCCGTCTCTGGGGTG GAGGTAGCACCACTGCAGAGCTTCATGCTGCGGGGTGGTG TTGTAGATGATCCAGTCGTAGCAGGAGCGCTGGGCGTGGT GCCTAAAAATGTCTTTCAGTAGCAAGCTGATTGCCAGGGG CAGGCCCTTGGTGTAAGTGTTTACAAAGCGGTTAAGCTGG GATGGGTGCATACGTGGGGATATGAGATGCATCTTGGACT GTATTTTTAGGTTGGCTATGTTCCCAGCCATATCCCTCCGG GGATTCATGTTGTGCAGAACCACCAGCACAGTGTATCCGG TGCACTTGGGAAATTTGTCATGTAGCTTAGAAGGAAATGC GTGGAAGAACTTGGAGACGCCCTTGTGACCTCCAAGATTTT CCATGCATTCGTCCATAATGATGGCAATGGGCCCACGGGC GGCGGCCTGGGCGAAGATATTTCTGGGATCACTAACGTCA TAGTTGTGTTCCAGGATGAGATCGTCATAGGCCATTTTTAC AAAGCGCGGGCGGAGGGTGCCAGACTGCGGTATAATGGTT CCATCCGGCCCAGGGGCGTAGTTACCCTCACAGATTTGCAT TTCCCACGCTTTGAGTTCAGATGGGGGGATCATGTCTACCT GCGGGGCGATGAAGAAAACGGTTTCCGGGGTAGGGGAGA TCAGCTGGGAAGAAAGCAGGTTCCTGAGCAGCTGCGACTT ACCGCAGCCGGTGGGCCCGTAAATCACACCTATTACCGGC TGCAACTGGTAGTTAAGAGAGCTGCAGCTGCCGTCATCCCT GAGCAGGGGGGCCACTTCGTTAAGCATGTCCCTGACTCGC ATGTTTTCCCTGACCAAATCCGCCAGAAGGCGCTCGCCGCC CAGCGATAGCAGTTCTTGCAAGGAAGCAAAGTTTTTCAAC GGTTTGAGACCGTCCGCCGTAGGCATGCTTTTGAGCGTTTG ACCAAGCAGTTCCAGGCGGTCCCACAGCTCGGTCACCTGC TCTACGGCATCTCGATCCAGCATATCTCCTCGTTTCGCGGG TTGGGGCGGCTTTCGCTGTACGGCAGTAGTCGGTGCTCGTC CAGACGGGCCAGGGTCATGTCTTTCCACGGGCGCAGGGTC CTCGTCAGCGTAGTCTGGGTCACGGTGAAGGGGTGCGCTC CGGGCTGCGCGCTGGCCAGGGTGCGCTTGAGGCTGGTCCT GCTGGTGCTGAAGCGCTGCCGGTCTTCGCCCTGCGCGTCGG CCAGGTAGCATTTGACCATGGTGTCATAGTCCAGCCCCTCC GCGGCGTGGCCCTTGGCGCGCAGCTTGCCCTTGGAGGAGG CGCCGCACGAGGGGCAGTGCAGACTTTTGAGGGCGTAGAG CTTGGGCGCGAGAAATACCGATTCCGGGGAGTAGGCATCC GCGCCGCAGGCCCCGCAGACGGTCTCGCATTCCACGAGCC AGGTGAGCTCTGGCCGTTCGGGGTCAAAAACCAGGTTTCC CCCATGCTTTTTGATGCGTTTCTTACCTCTGGTTTCCATGAG CCGGTGTCCACGCTCGGTGACGAAAAGGCTGTCCGTGTCC CCGTATACAGACTTGAGAGGCCTGTCCTCGAGCGGTGTTCC GCGGTCCTCCTCGTATAGAAACTCGGACCACTCTGAGACA AAGGCTCGCGTCCAGGCCAGCACGAAGGAGGCTAAGTGGG AGGGGTAGCGGTCGTTGTCCACTAGGGGGTCCACTCGCTC CAGGGTGTGAAGACACATGTCGCCCTCTTCGGCATCAAGG AAGGTGATTGGTTTGTAGGTGTAGGCCACGTGACCGGGTG TTCCTGAAGGGGGGCTATAAAAGGGGGTGGGGGCGCGTTC GTCCTCACTCTCTTCCGCATCGCTGTCTGCGAGGGCCAGCT GTTGGGGTGAGTACTCCCTCTGAAAAGCGGGCATGACTTCT GCGCTAAGATTGTCAGTTTCCAAAAACGAGGAGGATTTGA TATTCACCTGGCCCGCGGTGATGCCTTTGAGGGTGGCCGCA TCCATCTGGTCAGAAAAGACAATCTTTTTGTTGTCAAGCTT GGTGGCAAACGACCCGTAGAGGGCGTTGGACAGCAACTTG GCGATGGAGCGCAGGGTTTGGTTTTTGTCGCGATCGGCGC GCTCCTTGGCCGCGATGTTTAGCTGCACGTATTCGCGCGCA ACGCACCGCCATTCGGGAAAGACGGTGGTGCGCTCGTCGG GCACCAGGTGCACGCGCCAACCGCGGTTGTGCAGGGTGAC AAGGTCAACGCTGGTGGCTACCTCTCCGCGTAGGCGCTCGT TGGTCCAGCAGAGGCGGCCGCCCTTGCGCGAGCAGAATGG CGGTAGGGGGTCTAGCTGCGTCTCGTCCGGGGGGTCTGCG TCCACGGTAAAGACCCCGGGCAGCAGGCGCGCGTCGAAGT AGTCTATCTTGCATCCTTGCAAGTCTAGCGCCTGCTGCCAT GCGCGGGCGGCAAGCGCGCGCTCGTATGGGTTGAGTGGGG GACCCCATGGCATGGGGTGGGTGAGCGCGGAGGCGTACAT GCCGCAAATGTCGTAAACGTAGAGGGGCTCTCTGAGTATT CCAAGATATGTAGGGTAGCATCTTCCACCGCGGATGCTGG CGCGCACGTAATCGTATAGTTCGTGCGAGGGAGCGAGGAG GTCGGGACCGAGGTTGCTACGGGCGGGCTGCTCTGCTCGG AAGACTATCTGCCTGAAGATGGCATGTGAGTTGGATGATA TGGTTGGACGCTGGAAGACGTTGAAGCTGGCGTCTGTGAG ACCTACCGCGTCACGCACGAAGGAGGCGTAGGAGTCGCGC AGCTTGTTGACCAGCTCGGCGGTGACCTGCACGTCTAGGG CGCAGTAGTCCAGGGTTTCCTTGATGATGTCATACTTATCC TGTCCCTTTTTTTTCCACAGCTCGCGGTTGAGGACAAACTC TTCGCGGTCTTTCCAGTACTCTTGGATCGGAAACCCGTCGG CCTCCGAACGGTAAGAGCCTAGCATGTAGAACTGGTTGAC GGCCTGGTAGGCGCAGCATCCCTTTTCTACGGGTAGCGCGT ATGCCTGCGCGGCCTTCCGGAGCGAGGTGTGGGTGAGCGC AAAGGTGTCCCTGACCATGACTTTGAGGTACTGGTATTTGA AGTCAGTGTCGTCGCATCCGCCCTGCTCCCAGAGCAAAAA GTCCGTGCGCTTTTTGGAACGCGGATTTGGCAGGGCGAAG GTGACATCGTTGAAGAGTATCTTTCCCGCGCGAGGCATAA AGTTGCGTGTGATGCGGAAGGGTCCCGGCACCTCGGAACG GTTGTTAATTACCTGGGCGGCGAGCACGATCTCGTCAAAG CCGTTGATGTTGTGGCCCACAATGTAAAGTTCCAAGAAGC GCGGGATGCCCTTGATGGAAGGCAATTTTTTAAGTTCCTCG TAGGTGAGCTCTTCAGGGGAGCTGAGCCCGTGCTCTGAAA GGGCCCAGTCTGCAAGATGAGGGTTGGAAGCGACGAATGA GCTCCACAGGTCACGGGCCATTAGCATTTGCAGGTGGTCG CGAAAGGTCCTAAACTGGCGACCTATGGCCATTTTTTCTGG GGTGATGCAGTAGAAGGTAAGCGGGTCTTGTTCCCAGCGG TCCCATCCAAGGTTCGCGGCTAGGTCTCGCGCGGCAGTCAC TAGAGGCTCATCTCCGCCGAACTTCATGACCAGCATGAAG GGCACGAGCTGCTTCCCAAAGGCCCCCATCCAAGTATAGG TCTCTACATCGTAGGTGACAAAGAGACGCTCGGTGCGAGG ATGCGAGCCGATCGGGAAGAACTGGATCTCCCGCCACCAA TTGGAGGAGTGGCTATTGATGTGGTGAAAGTAGAAGTCCC TGCGACGGGCCGAACACTCGTGCTGGCTTTTGTAAAAACG TGCGCAGTACTGGCAGCGGTGCACGGGCTGTACATCCTGC ACGAGGTTGACCTGACGACCGCGCACAAGGAAGCAGAGTG GGAATTTGAGCCCCTCGCCTGGCGGGTTTGGCTGGTGGTCT TCTACTTCGGCTGCTTGTCCTTGACCGTCTGGCTGCTCGAG GGGAGTTACGGTGGATCGGACCACCACGCCGCGCGAGCCC AAAGTCCAGATGTCCGCGCGCGGCGGTCGGAGCTTGATGA CAACATCGCGCAGATGGGAGCTGTCCATGGTCTGGAGCTC CCGCGGCGTCAGGTCAGGCGGGAGCTCCTGCAGGTTTACC TCGCATAGACGGGTCAGGGCGCGGGCTAGATCCAGGTGAT ACCTAATTTCCAGGGGCTGGTTGGTGGCGGCGTCGATGGCT TGCAAGAGGCCGCATCCCCGCGGCGCGACTACGGTACCGC GCGGCGGGCGGTGGGCCGCGGGGGTGTCCTTGGATGATGC ATCTAAAAGCGGTGACGCGGGCGAGCCCCCGGAGGTAGGG GGGGCTCCGGACCCGCCGGGAGAGGGGGCAGGGGCACGT CGGCGCCGCGCGCGGGCAGGAGCTGGTGCTGCGCGCGTAG GTTGCTGGCGAACGCGACGACGCGGCGGTTGATCTCCTGA ATCTGGCGCCTCTGCGTGAAGACGACGGGCCCGGTGAGCT TGAACCTGAAAGAGAGTTCGACAGAATCAATTTCGGTGTC GTTGACGGCGGCCTGGCGCAAAATCTCCTGCACGTCTCCTG AGTTGTCTTGATAGGCGATCTCGGCCATGAACTGCTCGATC TCTTCCTCCTGGAGATCTCCGCGTCCGGCTCGCTCCACGGT GGCGGCGAGGTCGTTGGAAATGCGGGCCATGAGCTGCGAG AAGGCGTTGAGGCCTCCCTCGTTCCAGACGCGGCTGTAGA CCACGCCCCCTTCGGCATCGCGGGCGCGCATGACCACCTG CGCGAGATTGAGCTCCACGTGCCGGGCGAAGACGGCGTAG TTTCGCAGGCGCTGAAAGAGGTAGTTGAGGGTGGTGGCGG TGTGTTCTGCCACGAAGAAGTACATAACCCAGCGTCGCAA CGTGGATTCGTTGATATCCCCCAAGGCCTCAAGGCGCTCCA TGGCCTCGTAGAAGTCCACGGCGAAGTTGAAAAACTGGGA GTTGCGCGCCGACACGGTTAACTCCTCCTCCAGAAGACGG ATGAGCTCGGCGACAGTGTCGCGCACCTCGCGCTCAAAGG CTACAGGGGCCTCTTCTTCTTCTTCAATCTCCTCTTCCATAA GGGCCTCCCCTTCTTCTTCTTCTGGCGGCGGTGGGGGAGGG GGGACACGGCGGCGACGACGGCGCACCGGGAGGCGGTCG ACAAAGCGCTCGATCATCTCCCCGCGGCGACGGCGCATGG TCTCGGTGACGGCGCGGCCGTTCTCGCGGGGGCGCAGTTG GAAGACGCCGCCCGTCATGTCCCGGTTATGGGTTGGCGGG GGGCTGCCATGCGGCAGGGATACGGCGCTAACGATGCATC TCAACAATTGTTGTGTAGGTACTCCGCCGCCGAGGGACCTG AGCGAGTCCGCATCGACCGGATCGGAAAACCTCTCGAGAA AGGCGTCTAACCAGTCACAGTCGCAAGGTAGGCTGAGCAC CGTGGCGGGCGGCAGCGGGCGGCGGTCGGGGTTGTTTCTG GCGGAGGTGCTGCTGATGATGTAATTAAAGTAGGCGGTCT TGAGACGGCGGATGGTCGACAGAAGCACCATGTCCTTGGG TCCGGCCTGCTGAATGCGCAGGCGGTCGGCCATGCCCCAG GCTTCGTTTTGACATCGGCGCAGGTCTTTGTAGTAGTCTTG CATGAGCCTTTCTACCGGCACTTCTTCTTCTCCTTCCTCTTG TCCTGCATCTCTTGCATCTATCGCTGCGGCGGCGGCGGAGT TTGGCCGTAGGTGGCGCCCTCTTCCTCCCATGCGTGTGACC CCGAAGCCCCTCATCGGCTGAAGCAGGGCTAGGTCGGCGA CAACGCGCTCGGCTAATATGGCCTGCTGCACCTGCGTGAG GGTAGACTGGAAGTCATCCATGTCCACAAAGCGGTGGTAT GCGCCCGTGTTGATGGTGTAAGTGCAGTTGGCCATAACGG ACCAGTTAACGGTCTGGTGACCCGGCTGCGAGAGCTCGGT GTACCTGAGACGCGAGTAAGCCCTCGAGTCAAATACGTAG TCGTTGCAAGTCCGCACCAGGTACTGGTATCCCACCAAAA AGTGCGGCGGCGGCTGGCGGTAGAGGGGCCAGCGTAGGGT GGCCGGGGCTCCGGGGGCGAGATCTTCCAACATAAGGCGA TGATATCCGTAGATGTACCTGGACATCCAGGTGATGCCGG CGGCGGTGGTGGAGGCGCGCGGAAAGTCGCGGACGCGGTT CCAGATGTTGCGCAGCGGCAAAAAGTGCTCCATGGTCGGG ACGCTCTGGCCGGTCAGGCGCGCGCAATCGTTGACGCTCT AGCGTGCAAAAGGAGAGCCTGTAAGCGGGCACTCTTCCGT GGTCTGGTGGATAAATTCGCAAGGGTATCATGGCGGACGA CCGGGGTTCGAGCCCCGTATCCGGCCGTCCGCCGTGATCCA TGCGGTTACCGCCCGCGTGTCGAACCCAGGTGTGCGACGT CAGACAACGGGGGAGTGCTCCTTTTGGCTTCCTTCCAGGCG CGGCGGCTGCTGCGCTAGCTTTTTTGGCCACTGGCCGCGCG CAGCGTAAGCGGTTAGGCTGGAAAGCGAAAGCATTAAGTG GCTCGCTCCCTGTAGCCGGAGGGTTATTTTCCAAGGGTTGA GTCGCGGGACCCCCGGTTCGAGTCTCGGACCGGCCGGACT GCGGCGAACGGGGGTTTGCCTCCCCGTCATGCAAGACCCC GCTTGCAAATTCCTCCGGAAACAGGGACGAGCCCCTTTTTT GCTTTTCCCAGATGCATCCGGTGCTGCGGCAGATGCGCCCC CCTCCTCAGCAGCGGCAAGAGCAAGAGCAGCGGCAGACAT GCAGGGCACCCTCCCCTCCTCCTACCGCGTCAGGAGGGGC GACATCCGCGGTTGACGCGGCAGCAGATGGTGATTACGAA CCCCCGCGGCGCCGGGCCCGGCACTACCTGGACTTGGAGG AGGGCGAGGGCCTGGCGCGGCTAGGAGCGCCCTCTCCTGA GCGGCACCCAAGGGTGCAGCTGAAGCGTGATACGCGTGAG GCGTACGTGCCGCGGCAGAACCTGTTTCGCGACCGCGAGG GAGAGGAGCCCGAGGAGATGCGGGATCGAAAGTTCCACG CAGGGCGCGAGCTGCGGCATGGCCTGAATCGCGAGCGGTT GCTGCGCGAGGAGGACTTTGAGCCCGACGCGCGAACCGGG ATTAGTCCCGCGCGCGCACACGTGGCGGCCGCCGACCTGG TAACCGCATACGAGCAGACGGTGAACCAGGAGATTAACTT TCAAAAAAGCTTTAACAACCACGTGCGTACGCTTGTGGCG CGCGAGGAGGTGGCTATAGGACTGATGCATCTGTGGGACT TTGTAAGCGCGCTGGAGCAAAACCCAAATAGCAAGCCGCT CATGGCGCAGCTGTTCCTTATAGTGCAGCACAGCAGGGAC AACGAGGCATTCAGGGATGCGCTGCTAAACATAGTAGAGC CCGAGGGCCGCTGGCTGCTCGATTTGATAAACATCCTGCA GAGCATAGTGGTGCAGGAGCGCAGCTTGAGCCTGGCTGAC AAGGTGGCCGCCATCAACTATTCCATGCTTAGCCTGGGCA AGTTTTACGCCCGCAAGATATACCATACCCCTTACGTTCCC ATAGACAAGGAGGTAAAGATCGAGGGGTTCTACATGCGCA TGGCGCTGAAGGTGCTTACCTTGAGCGACGACCTGGGCGT TTATCGCAACGAGCGCATCCACAAGGCCGTGAGCGTGAGC CGGCGGCGCGAGCTCAGCGACCGCGAGCTGATGCACAGCC TGCAAAGGGCCCTGGCTGGCACGGGCAGCGGCGATAGAGA GGCCGAGTCCTACTTTGACGCGGGCGCTGACCTGCGCTGG GCCCCAAGCCGACGCGCCCTGGAGGCAGCTGGGGCCGGAC CTGGGCTGGCGGTGGCACCCGCGCGCGCTGGCAACGTCGG CGGCGTGGAGGAATATGACGAGGACGATGAGTACGAGCC AGAGGACGGCGAGTACTAAGCGGTGATGTTTCTGATCAGA TGATGCAAGACGCAACGGACCCGGCGGTGCGGGCGGCGCT GCAGAGCCAGCCGTCCGGCCTTAACTCCACGGACGACTGG CGCCAGGTCATGGACCGCATCATGTCGCTGACTGCGCGCA ATCCTGACGCGTTCCGGCAGCAGCCGCAGGCCAACCGGCT CTCCGCAATTCTGGAAGCGGTGGTCCCGGCGCGCGCAAAC CCCACGCACGAGAAGGTGCTGGCGATCGTAAACGCGCTGG CCGAAAACAGGGCCATCCGGCCCGACGAGGCCGGCCTGGT CTACGACGCGCTGCTTCAGCGCGTGGCTCGTTACAACAGC GGCAACGTGCAGACCAACCTGGACCGGCTGGTGGGGGATG TGCGCGAGGCCGTGGCGCAGCGTGAGCGCGCGCAGCAGCA GGGCAACCTGGGCTCCATGGTTGCACTAAACGCCTTCCTGA GTACACAGCCCGCCAACGTGCCGCGGGGACAGGAGGACTA CACCAACTTTGTGAGCGCACTGCGGCTAATGGTGACTGAG ACACCGCAAAGTGAGGTGTACCAGTCTGGGCCAGACTATT TTTTCCAGACCAGTAGACAAGGCCTGCAGACCGTAAACCT GAGCCAGGCTTTCAAAAACTTGCAGGGGCTGTGGGGGGTG CGGGCTCCCACAGGCGACCGCGCGACCGTGTCTAGCTTGC TGACGCCCAACTCGCGCCTGTTGCTGCTGCTAATAGCGCCC TTCACGGACAGTGGCAGCGTGTCCCGGGACACATACCTAG GTCACTTGCTGACACTGTACCGCGAGGCCATAGGTCAGGC GCATGTGGACGAGCATACTTTCCAGGAGATTACAAGTGTC AGCCGCGCGCTGGGGCAGGAGGACACGGGCAGCCTGGAG GCAACCCTAAACTACCTGCTGACCAACCGGCGGCAGAAGA TCCCCTCGTTGCACAGTTTAAACAGCGAGGAGGAGCGCAT TTTGCGCTACGTGCAGCAGAGCGTGAGCCTTAACCTGATGC GCGACGGGGTAACGCCCAGCGTGGCGCTGGACATGACCGC GCGCAACATGGAACCGGGCATGTATGCCTCAAACCGGCCG TTTATCAACCGCCTAATGGACTACTTGCATCGCGCGGCCGC CGTGAACCCCGAGTATTTCACCAATGCCATCTTGAACCCGC ACTGGCTACCGCCCCCTGGTTTCTACACCGGGGGATTCGAG GTGCCCGAGGGTAACGATGGATTCCTCTGGGACGACATAG ACGACAGCGTGTTTTCCCCGCAACCGCAGACCCTGCTAGA GTTGCAACAGCGCGAGCAGGCAGAGGCGGCGCTGCGAAA GGAAAGCTTCCGCAGGCCAAGCAGCTTGTCCGATCTAGGC GCTGCGGCCCCGCGGTCAGATGCTAGTAGCCCATTTCCAA GCTTGATAGGGTCTCTTACCAGCACTCGCACCACCCGCCCG CGCCTGCTGGGCGAGGAGGAGTACCTAAACAACTCGCTGC TGCAGCCGCAGCGCGAAAAAAACCTGCCTCCGGCATTTCC CAACAACGGGATAGAGAGCCTAGTGGACAAGATGAGTAG ATGGAAGACGTACGCGCAGGAGCACAGGGACGTGCCAGG CCCGCGCCCGCCCACCCGTCGTCAAAGGCACGACCGTCAG CGGGGTCTGGTGTGGGAGGACGATGACTCGGCAGACGACA GCAGCGTCCTGGATTTGGGAGGGAGTGGCAACCCGTTTGC GCACCTTCGCCCCAGGCTGGGGAGAATGTTTTAAAAAAAA AAAAGCATGATGCAAAATAAAAAACTCACCAAGGCCATGG CACCGAGCGTTGGTTTTCTTGTATTCCCCTTAGTATGCGGC GCGCGGCGATGTATGAGGAAGGTCCTCCTCCCTCCTACGA GAGTGTGGTGAGCGCGGCGCCAGTGGCGGCGGCGCTGGGT TCTCCCTTCGATGCTCCCCTGGACCCGCCGTTTGTGCCTCC GCGGTACCTGCGGCCTACCGGGGGGAGAAACAGCATCCGT TACTCTGAGTTGGCACCCCTATTCGACACCACCCGTGTGTA CCTGGTGGACAACAAGTCAACGGATGTGGCATCCCTGAAC TACCAGAACGACCACAGCAACTTTCTGACCACGGTCATTC AAAACAATGACTACAGCCCGGGGGAGGCAAGCACACAGA CCATCAATCTTGACGACCGGTCGCACTGGGGCGGCGACCT GAAAACCATCCTGCATACCAACATGCCAAATGTGAACGAG TTCATGTTTACCAATAAGTTTAAGGCGCGGGTGATGGTGTC GCGCTTGCCTACTAAGGACAATCAGGTGGAGCTGAAATAC GAGTGGGTGGAGTTCACGCTGCCCGAGGGCAACTACTCCG AGACCATGACCATAGACCTTATGAACAACGCGATCGTGGA GCACTACTTGAAAGTGGGCAGACAGAACGGGGTTCTGGAA AGCGACATCGGGGTAAAGTTTGACACCCGCAACTTCAGAC TGGGGTTTGACCCCGTCACTGGTCTTGTCATGCCTGGGGTA TATACAAACGAAGCCTTCCATCCAGACATCATTTTGCTGCC AGGATGCGGGGTGGACTTCACCCACAGCCGCCTGAGCAAC TTGTTGGGCATCCGCAAGCGGCAACCCTTCCAGGAGGGCT TTAGGATCACCTACGATGATCTGGAGGGTGGTAACATTCCC GCACTGTTGGATGTGGACGCCTACCAGGCGAGCTTGAAAG ATGACACCGAACAGGGCGGGGGTGGCGCAGGCGGCAGCA ACAGCAGTGGCAGCGGCGCGGAAGAGAACTCCAACGCGG CAGCCGCGGCAATGCAGCCGGTGGAGGACATGAACGATCA TGCCATTCGCGGCGACACCTTTGCCACACGGGCTGAGGAG AAGCGCGCTGAGGCCGAAGCAGCGGCCGAAGCTGCCGCCC CCGCTGCGCAACCCGAGGTCGAGAAGCCTCAGAAGAAACC GGTGATCAAACCCCTGACAGAGGACAGCAAGAAACGCAGT TACAACCTAATAAGCAATGACAGCACCTTCACCCAGTACC GCAGCTGGTACCTTGCATACAACTACGGCGACCCTCAGAC CGGAATCCGCTCATGGACCCTGCTTTGCACTCCTGACGTAA CCTGCGGCTCGGAGCAGGTCTACTGGTCGTTGCCAGACAT GATGCAAGACCCCGTGACCTTCCGCTCCACGCGCCAGATC AGCAACTTTCCGGTGGTGGGCGCCGAGCTGTTGCCCGTGC ACTCCAAGAGCTTCTACAACGACCAGGCCGTCTACTCCCA ACTCATCCGCCAGTTTACCTCTCTGACCCACGTGTTCAATC GCTTTCCCGAGAACCAGATTTTGGCGCGCCCGCCAGCCCCC ACCATCACCACCGTCAGTGAAAACGTTCCTGCTCTCACAGA TCACGGGACGCTACCGCTGCGCAACAGCATCGGAGGAGTC CAGCGAGTGACCATTACTGACGCCAGACGCCGCACCTGCC CCTACGTTTACAAGGCCCTGGGCATAGTCTCGCCGCGCGTC CTATCGAGCCGCACTTTTTGAGCAAGCATGTCCATCCTTAT ATCGCCCAGCAATAACACAGGCTGGGGCCTGCGCTTCCCA AGCAAGATGTTTGGCGGGGCCAAGAAGCGCTCCGACCAAC ACCCAGTGCGCGTGCGCGGGCACTACCGCGCGCCCTGGGG CGCGCACAAACGCGGCCGCACTGGGCGCACCACCGTCGAT GACGCCATCGACGCGGTGGTGGAGGAGGCGCGCAACTACA CGCCCACGCCGCCACCAGTGTCCACAGTGGACGCGGCCAT TCAGACCGTGGTGCGCGGAGCCCGGCGCTATGCTAAAATG AAGAGACGGCGGAGGCGCGTAGCACGTCGCCACCGCCGCC GACCCGGCACTGCCGCCCAACGCGCGGCGGCGGCCCTGCT TAACCGCGCACGTCGCACCGGCCGACGGGCGGCCATGCGG GCCGCTCGAAGGCTGGCCGCGGGTATTGTCACTGTGCCCCC CAGGTCCAGGCGACGAGCGGCCGCCGCAGCAGCCGCGGCC ATTAGTGCTATGACTCAGGGTCGCAGGGGCAACGTGTATT GGGTGCGCGACTCGGTTAGCGGCCTGCGCGTGCCCGTGCG CACCCGCCCCCCGCGCAACTAGATTGCAAGAAAAAACTAC TTAGACTCGTACTGTTGTATGTATCCAGCGGCGGCGGCGCG CAACGAAGCTATGTCCAAGCGCAAAATCAAAGAAGAGATG CTCCAGGTCATCGCGCCGGAGATCTATGGCCCCCCGAAGA AGGAAGAGCAGGATTACAAGCCCCGAAAGCTAAAGCGGG TCAAAAAGAAAAAGAAAGATGATGATGATGAACTTGACG ACGAGGTGGAACTGCTGCACGCTACCGCGCCCAGGCGACG GGTACAGTGGAAAGGTCGACGCGTAAAACGTGTTTTGCGA CCCGGCACCACCGTAGTCTTTACGCCCGGTGAGCGCTCCAC CCGCACCTACAAGCGCGTGTATGATGAGGTGTACGGCGAC GAGGACCTGCTTGAGCAGGCCAACGAGCGCCTCGGGGAGT TTGCCTACGGAAAGCGGCATAAGGACATGCTGGCGTTGCC GCTGGACGAGGGCAACCCAACACCTAGCCTAAAGCCCGTA ACACTGCAGCAGGTGCTGCCCGCGCTTGCACCGTCCGAAG AAAAGCGCGGCCTAAAGCGCGAGTCTGGTGACTTGGCACC CACCGTGCAGCTGATGGTACCCAAGCGCCAGCGACTGGAA GATGTCTTGGAAAAAATGACCGTGGAACCTGGGCTGGAGC CCGAGGTCCGCGTGCGGCCAATCAAGCAGGTGGCGCCGGG ACTGGGCGTGCAGACCGTGGACGTTCAGATACCCACTACC AGTAGCACCAGTATTGCCACCGCCACAGAGGGCATGGAGA CACAAACGTCCCCGGTTGCCTCAGCGGTGGCGGATGCCGC GGTGCAGGCGGTCGCTGCGGCCGCGTCCAAGACCTCTACG GAGGTGCAAACGGACCCGTGGATGTTTCGCGTTTCAGCCC CCCGGCGCCCGCGCCGTTCGAGGAAGTACGGCGCCGCCAG CGCGCTACTGCCCGAATATGCCCTACATCCTTCCATTGCGC CTACCCCCGGCTATCGTGGCTACACCTACCGCCCCAGAAG ACGAGCAACTACCCGACGCCGAACCACCACTGGAACCCGC CGCCGCCGTCGCCGTCGCCAGCCCGTGCTGGCCCCGATTTC CGTGCGCAGGGTGGCTCGCGAAGGAGGCAGGACCCTGGTG CTGCCAACAGCGCGCTACCACCCCAGCATCGTTTAAAAGC CGGTCTTTGTGGTTCTTGCAGATATGGCCCTCACCTGCCGC CTCCGTTTCCCGGTGCCGGGATTCCGAGGAAGAATGCACC GTAGGAGGGGCATGGCCGGCCACGGCCTGACGGGCGGCAT GCGTCGTGCGCACCACCGGCGGCGGCGCGCGTCGCACCGT CGCATGCGCGGCGGTATCCTGCCCCTCCTTATTCCACTGAT CGCCGCGGCGATTGGCGCCGTGCCCGGAATTGCATCCGTG GCCTTGCAGGCGCAGAGACACTGATTAAAAACAAGTTGCA TGTGGAAAAATCAAAATAAAAAGTCTGGACTCTCACGCTC GCTTGGTCCTGTAACTATTTTGTAGAATGGAAGACATCAAC TTTGCGTCTCTGGCCCCGCGACACGGCTCGCGCCCGTTCAT GGGAAACTGGCAAGATATCGGCACCAGCAATATGAGCGGT GGCGCCTTCAGCTGGGGCTCGCTGTGGAGCGGCATTAAAA ATTTCGGTTCCACCGTTAAGAACTATGGCAGCAAGGCCTG GAACAGCAGCACAGGCCAGATGCTGAGGGATAAGTTGAA AGAGCAAAATTTCCAACAAAAGGTGGTAGATGGCCTGGCC TCTGGCATTAGCGGGGTGGTGGACCTGGCCAACCAGGCAG TGCAAAATAAGATTAACAGTAAGCTTGATCCCCGCCCTCCC GTAGAGGAGCCTCCACCGGCCGTGGAGACAGTGTCTCCAG AGGGGCGTGGCGAAAAGCGTCCGCGCCCCGACAGGGAAG AAACTCTGGTGACGCAAATAGACGAGCCTCCCTCGTACGA GGAGGCACTAAAGCAAGGCCTGCCCACCACCCGTCCCATC GCGCCCATGGCTACCGGAGTGCTGGGCCAGCACACACCCG TAACGCTGGACCTGCCTCCCCCCGCCGACACCCAGCAGAA ACCTGTGCTGCCAGGCCCGACCGCCGTTGTTGTAACCCGTC CTAGCCGCGCGTCCCTGCGCCGCGCCGCCAGCGGTCCGCG ATCGTTGCGGCCCGTAGCCAGTGGCAACTGGCAAAGCACA CTGAACAGCATCGTGGGTCTGGGGGTGCAATCCCTGAAGC GCCGACGATGCTTCTGATAGCTAACGTGTCGTATGTGTGTC ATGTATGCGTCCATGTCGCCGCCAGAGGAGCTGCTGAGCC GCCGCGCGCCCGCTTTCCAAGATGGCTACCCCTTCGATGAT GCCGCAGTGGTCTTACATGCACATCTCGGGCCAGGACGCC TCGGAGTACCTGAGCCCCGGGCTGGTGCAGTTTGCCCGCG CCACCGAGACGTACTTCAGCCTGAATAACAAGTTTAGAAA CCCCACGGTGGCGCCTACGCACGACGTGACCACAGACCGG TCCCAGCGTTTGACGCTGCGGTTCATCCCTGTGGACCGTGA GGATACTGCGTACTCGTACAAGGCGCGGTTCACCCTAGCT GTGGGTGATAACCGTGTGCTGGACATGGCTTCCACGTACTT TGACATCCGCGGCGTGCTGGACAGGGGCCCTACTTTTAAG CCCTACTCTGGCACTGCCTACAACGCCCTGGCTCCCAAGGG TGCCCCAAATCCTTGCGAATGGGATGAAGCTGCTACTGCTC TTGAAATAAACCTAGAAGAAGAGGACGATGACAACGAAG ACGAAGTAGACGAGCAAGCTGAGCAGCAAAAAACTCACG TATTTGGGCAGGCGCCTTATTCTGGTATAAATATTACAAAG GAGGGTATTCAAATAGGTGTCGAAGGTCAAACACCTAAAT ATGCCGATAAAACATTTCAACCTGAACCTCAAATAGGAGA ATCTCAGTGGTACGAAACAGAAATTAATCATGCAGCTGGG AGAGTCCTAAAAAAGACTACCCCAATGAAACCATGTTACG GTTCATATGCAAAACCCACAAATGAAAATGGAGGGCAAGG CATTCTTGTAAAGCAACAAAATGGAAAGCTAGAAAGTCAA GTGGAAATGCAATTTTTCTCAACTACTGAGGCAGCCGCAG GCAATGGTGATAACTTGACTCCTAAAGTGGTATTGTACAGT GAAGATGTAGATATAGAAACCCCAGACACTCATATTTCTT ACATGCCCACTATTAAGGAAGGTAACTCACGAGAACTAAT GGGCCAACAATCTATGCCCAACAGGCCTAATTACATTGCTT TTAGGGACAATTTTATTGGTCTAATGTATTACAACAGCACG GGTAATATGGGTGTTCTGGCGGGCCAAGCATCGCAGTTGA ATGCTGTTGTAGATTTGCAAGACAGAAACACAGAGCTTTC ATACCAGCTTTTGCTTGATTCCATTGGTGATAGAACCAGGT ACTTTTCTATGTGGAATCAGGCTGTTGACAGCTATGATCCA GATGTTAGAATTATTGAAAATCATGGAACTGAAGATGAAC TTCCAAATTACTGCTTTCCACTGGGAGGTGTGATTAATACA GAGACTCTTACCAAGGTAAAACCTAAAACAGGTCAGGAAA ATGGATGGGAAAAAGATGCTACAGAATTTTCAGATAAAAA TGAAATAAGAGTTGGAAATAATTTTGCCATGGAAATCAAT CTAAATGCCAACCTGTGGAGAAATTTCCTGTACTCCAACAT AGCGCTGTATTTGCCCGACAAGCTAAAGTACAGTCCTTCCA ACGTAAAAATTTCTGATAACCCAAACACCTACGACTACAT GAACAAGCGAGTGGTGGCTCCCGGGCTAGTGGACTGCTAC ATTAACCTTGGAGCACGCTGGTCCCTTGACTATATGGACAA CGTCAACCCATTTAACCACCACCGCAATGCTGGCCTGCGCT ACCGCTCAATGTTGCTGGGCAATGGTCGCTATGTGCCCTTC CACATCCAGGTGCCTCAGAAGTTCTTTGCCATTAAAAACCT CCTTCTCCTGCCGGGCTCATACACCTACGAGTGGAACTTCA GGAAGGATGTTAACATGGTTCTGCAGAGCTCCCTAGGAAA TGACCTAAGGGTTGACGGAGCCAGCATTAAGTTTGATAGC ATTTGCCTTTACGCCACCTTCTTCCCCATGGCCCACAACAC CGCCTCCACGCTTGAGGCCATGCTTAGAAACGACACCAAC GACCAGTCCTTTAACGACTATCTCTCCGCCGCCAACATGCT CTACCCTATACCCGCCAACGCTACCAACGTGCCCATATCCA TCCCCTCCCGCAACTGGGCGGCTTTCCGCGGCTGGGCCTTC ACGCGCCTTAAGACTAAGGAAACCCCATCACTGGGCTCGG GCTACGACCCTTATTACACCTACTCTGGCTCTATACCCTAC CTAGATGGAACCTTTTACCTCAACCACACCTTTAAGAAGGT GGCCATTACCTTTGACTCTTCTGTCAGCTGGCCTGGCAATG ACCGCCTGCTTACCCCCAACGAGTTTGAAATTAAGCGCTCA GTTGACGGGGAGGGTTACAACGTTGCCCAGTGTAACATGA CCAAAGACTGGTTCCTGGTACAAATGCTAGCTAACTATAA CATTGGCTACCAGGGCTTCTATATCCCAGAGAGCTACAAG GACCGCATGTACTCCTTCTTTAGAAACTTCCAGCCCATGAG CCGTCAGGTGGTGGATGATACTAAATACAAGGACTACCAA CAGGTGGGCATCCTACACCAACACAACAACTCTGGATTTG TTGGCTACCTTGCCCCCACCATGCGCGAAGGACAGGCCTA CCCTGCTAACTTCCCCTATCCGCTTATAGGCAAGACCGCAG TTGACAGCATTACCCAGAAAAAGTTTCTTTGCGATCGCACC CTTTGGCGCATCCCATTCTCCAGTAACTTTATGTCCATGGG CGCACTCACAGACCTGGGCCAAAACCTTCTCTACGCCAACT CCGCCCACGCGCTAGACATGACTTTTGAGGTGGATCCCATG GACGAGCCCACCCTTCTTTATGTTTTGTTTGAAGTCTTTGAC GTGGTCCGTGTGCACCAGCCGCACCGCGGCGTCATCGAAA CCGTGTACCTGCGCACGCCCTTCTCGGCCGGCAACGCCACA ACATAAAGAAGCAAGCAACATCAACAACAGCTGCCGCCAT GGGCTCCAGTGAGCAGGAACTGAAAGCCATTGTCAAAGAT CTTGGTTGTGGGCCATATTTTTTGGGCACCTATGACAAGCG CTTTCCAGGCTTTGTTTCTCCACACAAGCTCGCCTGCGCCA TAGTCAATACGGCCGGTCGCGAGACTGGGGGCGTACACTG GATGGCCTTTGCCTGGAACCCGCACTCAAAAACATGCTAC CTCTTTGAGCCCTTTGGCTTTTCTGACCAGCGACTCAAGCA GGTTTACCAGTTTGAGTACGAGTCACTCCTGCGCCGTAGCG CCATTGCTTCTTCCCCCGACCGCTGTATAACGCTGGAAAAG TCCACCCAAAGCGTACAGGGGCCCAACTCGGCCGCCTGTG GACTATTCTGCTGCATGTTTCTCCACGCCTTTGCCAACTGG CCCCAAACTCCCATGGATCACAACCCCACCATGAACCTTAT TACCGGGGTACCCAACTCCATGCTCAACAGTCCCCAGGTA CAGCCCACCCTGCGTCGCAACCAGGAACAGCTCTACAGCT TCCTGGAGCGCCACTCGCCCTACTTCCGCAGCCACAGTGCG CAGATTAGGAGCGCCACTTCTTTTTGTCACTTGAAAAACAT GTAAAAATAATGTACTAGAGACACTTTCAATAAAGGCAAA TGCTTTTATTTGTACACTCTCGGGTGATTATTTACCCCCACC CTTGCCGTCTGCGCCGTTTAAAAATCAAAGGGGTTCTGCCG CGCATCGCTATGCGCCACTGGCAGGGACACGTTGCGATAC TGGTGTTTAGTGCTCCACTTAAACTCAGGCACAACCATCCG CGGCAGCTCGGTGAAGTTTTCACTCCACAGGCTGCGCACC ATCACCAACGCGTTTAGCAGGTCGGGCGCCGATATCTTGA AGTCGCAGTTGGGGCCTCCGCCCTGCGCGCGCGAGTTGCG ATACACAGGGTTGCAGCACTGGAACACTATCAGCGCCGGG TGGTGCACGCTGGCCAGCACGCTCTTGTCGGAGATCAGAT CCGCGTCCAGGTCCTCCGCGTTGCTCAGGGCGAACGGAGT CAACTTTGGTAGCTGCCTTCCCAAAAAGGGCGCGTGCCCA GGCTTTGAGTTGCACTCGCACCGTAGTGGCATCAAAAGGT GACCGTGCCCGGTCTGGGCGTTAGGATACAGCGCCTGCAT AAAAGCCTTGATCTGCTTAAAAGCCACCTGAGCCTTTGCGC CTTCAGAGAAGAACATGCCGCAAGACTTGCCGGAAAACTG ATTGGCCGGACAGGCCGCGTCGTGCACGCAGCACCTTGCG TCGGTGTTGGAGATCTGCACCACATTTCGGCCCCACCGGTT CTTCACGATCTTGGCCTTGCTAGACTGCTCCTTCAGCGCGC GCTGCCCGTTTTCGCTCGTCACATCCATTTCAATCACGTGC TCCTTATTTATCATAATGCTTCCGTGTAGACACTTAAGCTC GCCTTCGATCTCAGCGCAGCGGTGCAGCCACAACGCGCAG CCCGTGGGCTCGTGATGCTTGTAGGTCACCTCTGCAAACGA CTGCAGGTACGCCTGCAGGAATCGCCCCATCATCGTCACA AAGGTCTTGTTGCTGGTGAAGGTCAGCTGCAACCCGCGGT GCTCCTCGTTCAGCCAGGTCTTGCATACGGCCGCCAGAGCT TCCACTTGGTCAGGCAGTAGTTTGAAGTTCGCCTTTAGATC GTTATCCACGTGGTACTTGTCCATCAGCGCGCGCGCAGCCT CCATGCCCTTCTCCCACGCAGACACGATCGGCACACTCAGC GGGTTCATCACCGTAATTTCACTTTCCGCTTCGCTGGGCTC TTCCTCTTCCTCTTGCGTCCGCATACCACGCGCCACTGGGT CGTCTTCATTCAGCCGCCGCACTGTGCGCTTACCTCCTTTG CCATGCTTGATTAGCACCGGTGGGTTGCTGAAACCCACCAT TTGTAGCGCCACATCTTCTCTTTCTTCCTCGCTGTCCACGAT TACCTCTGGTGATGGCGGGCGCTCGGGCTTGGGAGAAGGG CGCTTCTTTTTCTTCTTGGGCGCAATGGCCAAATCCGCCGC CGAGGTCGATGGCCGCGGGCTGGGTGTGCGCGGCACCAGC GCGTCTTGTGATGAGTCTTCCTCGTCCTCGGACTCGATACG CCGCCTCATCCGCTTTTTTGGGGGCGCCCGGGGAGGCGGC GGCGACGGGGACGGGGACGACACGTCCTCCATGGTTGGGG GACGTCGCGCCGCACCGCGTCCGCGCTCGGGGGTGGTTTC GCGCTGCTCCTCTTCCCGACTGGCCATTTCCTTCTCCTATAG GCAGAAAAAGATCATGGAGTCAGTCGAGAAGAAGGACAG CCTAACCGCCCCCTCTGAGTTCGCCACCACCGCCTCCACCG ATGCCGCCAACGCGCCTACCACCTTCCCCGTCGAGGCACCC CCGCTTGAGGAGGAGGAAGTGATTATCGAGCAGGACCCAG GTTTTGTAAGCGAAGACGACGAGGACCGCTCAGTACCAAC AGAGGATAAAAAGCAAGACCAGGACAACGCAGAGGCAAA CGAGGAACAAGTCGGGCGGGGGGACGAAAGGCATGGCGA CTACCTAGATGTGGGAGACGACGTGCTGTTGAAGCATCTG CAGCGCCAGTGCGCCATTATCTGCGACGCGTTGCAAGAGC GCAGCGATGTGCCCCTCGCCATAGCGGATGTCAGCCTTGCC TACGAACGCCACCTATTCTCACCGCGCGTACCCCCCAAACG CCAAGAAAACGGCACATGCGAGCCCAACCCGCGCCTCAAC TTCTACCCCGTATTTGCCGTGCCAGAGGTGCTTGCCACCTA TCACATCTTTTTCCAAAACTGCAAGATACCCCTATCCTGCC GTGCCAACCGCAGCCGAGCGGACAAGCAGCTGGCCTTGCG GCAGGGCGCTGTCATACCTGATATCGCCTCGCTCAACGAA GTGCCAAAAATCTTTGAGGGTCTTGGACGCGACGAGAAGC GCGCGGCAAACGCTCTGCAACAGGAAAACAGCGAAAATG AAAGTCACTCTGGAGTGTTGGTGGAACTCGAGGGTGACAA CGCGCGCCTAGCCGTACTAAAACGCAGCATCGAGGTCACC CACTTTGCCTACCCGGCACTTAACCTACCCCCCAAGGTCAT GAGCACAGTCATGAGTGAGCTGATCGTGCGCCGTGCGCAG CCCCTGGAGAGGGATGCAAATTTGCAAGAACAAACAGAGG AGGGCCTACCCGCAGTTGGCGACGAGCAGCTAGCGCGCTG GCTTCAAACGCGCGAGCCTGCCGACTTGGAGGAGCGACGC AAACTAATGATGGCCGCAGTGCTCGTTACCGTGGAGCTTG AGTGCATGCAGCGGTTCTTTGCTGACCCGGAGATGCAGCG CAAGCTAGAGGAAACATTGCACTACACCTTTCGACAGGGC TACGTACGCCAGGCCTGCAAGATCTCCAACGTGGAGCTCT GCAACCTGGTCTCCTACCTTGGAATTTTGCACGAAAACCGC CTTGGGCAAAACGTGCTTCATTCCACGCTCAAGGGCGAGG CGCGCCGCGACTACGTCCGCGACTGCGTTTACTTATTTCTA TGCTACACCTGGCAGACGGCCATGGGCGTTTGGCAGCAGT GCTTGGAGGAGTGCAACCTCAAGGAGCTGCAGAAACTGCT AAAGCAAAACTTGAAGGACCTATGGACGGCCTTCAACGAG CGCTCCGTGGCCGCGCACCTGGCGGACATCATTTTCCCCGA ACGCCTGCTTAAAACCCTGCAACAGGGTCTGCCAGACTTC ACCAGTCAAAGCATGTTGCAGAACTTTAGGAACTTTATCCT AGAGCGCTCAGGAATCTTGCCCGCCACCTGCTGTGCACTTC CTAGCGACTTTGTGCCCATTAAGTACCGCGAATGCCCTCCG CCGCTTTGGGGCCACTGCTACCTTCTGCAGCTAGCCAACTA CCTTGCCTACCACTCTGACATAATGGAAGACGTGAGCGGT GACGGTCTACTGGAGTGTCACTGTCGCTGCAACCTATGCAC CCCGCACCGCTCCCTGGTTTGCAATTCGCAGCTGCTTAACG AAAGTCAAATTATCGGTACCTTTGAGCTGCAGGGTCCCTCG CCTGACGAAAAGTCCGCGGCTCCGGGGTTGAAACTCACTC CGGGGCTGTGGACGTCGGCTTACCTTCGCAAATTTGTACCT GAGGACTACCACGCCCACGAGATTAGGTTCTACGAAGACC AATCCCGCCCGCCTAATGCGGAGCTTACCGCCTGCGTCATT ACCCAGGGCCACATTCTTGGCCAATTGCAAGCCATCAACA AAGCCCGCCAAGAGTTTCTGCTACGAAAGGGACGGGGGGT TTACTTGGACCCCCAGTCCGGCGAGGAGCTCAACCCAATC CCCCCGCCGCCGCAGCCCTATCAGCAGCAGCCGCGGGCCC TTGCTTCCCAGGATGGCACCCAAAAAGAAGCTGCAGCTGC CGCCGCCACCCACGGACGAGGAGGAATACTGGGACAGTCA GGCAGAGGAGGTTTTGGACGAGGAGGAGGAGGACATGAT GGAAGACTGGGAGAGCCTAGACGAGGAAGCTTCCGAGGTC GAAGAGGTGTCAGACGAAACACCGTCACCCTCGGTCGCAT TCCCCTCGCCGGCGCCCCAGAAATCGGCAACCGGTTCCAG CATGGCTACAACCTCCGCTCCTCAGGCGCCGCCGGCACTGC CCGTTCGCCGACCCAACCGTAGATGGGACACCACTGGAAC CAGGGCCGGTAAGTCCAAGCAGCCGCCGCCGTTAGCCCAA GAGCAACAACAGCGCCAAGGCTACCGCTCATGGCGCGGGC ACAAGAACGCCATAGTTGCTTGCTTGCAAGACTGTGGGGG CAACATCTCCTTCGCCCGCCGCTTTCTTCTCTACCATCACG GCGTGGCCTTCCCCCGTAACATCCTGCATTACTACCGTCAT CTCTACAGCCCATACTGCACCGGCGGCAGCGGCAGCAACA GCAGCGGCCACACAGAAGCAAAGGCGACCGGATAGCAAG ACTCTGACAAAGCCCAAGAAATCCACAGCGGCGGCAGCAG CAGGAGGAGGAGCGCTGCGTCTGGCGCCCAACGAACCCGT ATCGACCCGCGAGCTTAGAAACAGGATTTTTCCCACTCTGT ATGCTATATTTCAACAGAGCAGGGGCCAAGAACAAGAGCT GAAAATAAAAAACAGGTCTCTGCGATCCCTCACCCGCAGC TGCCTGTATCACAAAAGCGAAGATCAGCTTCGGCGCACGC TGGAAGACGCGGAGGCTCTCTTCAGTAAATACTGCGCGCT GACTCTTAAGGACTAGTTTCGCGCCCTTTCTCAAATTTAAG CGCGAAAACTACGTCATCTCCAGCGGCCACACCCGGCGCC AGCACCTGTTGTCAGCGCCATTATGAGCAAGGAAATTCCC ACGCCCTACATGTGGAGTTACCAGCCACAAATGGGACTTG CGGCTGGAGCTGCCCAAGACTACTCAACCCGAATAAACTA CATGAGCGCGGGACCCCACATGATATCCCGGGTCAACGGA ATACGCGCCCACCGAAACCGAATTCTCCTGGAACAGGCGG CTATTACCACCACACCTCGTAATAACCTTAATCCCCGTAGT TGGCCCGCTGCCCTGGTGTACCAGGAAAGTCCCGCTCCCAC CACTGTGGTACTTCCCAGAGACGCCCAGGCCGAAGTTCAG ATGACTAACTCAGGGGCGCAGCTTGCGGGCGGCTTTCGTC ACAGGGTGCGGTCGCCCGGGCAGGGTATAACTCACCTGAC AATCAGAGGGCGAGGTATTCAGCTCAACGACGAGTCGGTG AGCTCCTCGCTTGGTCTCCGTCCGGACGGGACATTTCAGAT CGGCGGCGCCGGCCGCTCTTCATTCACGCCTCGTCAGGCAA TCCTAACTCTGCAGACCTCGTCCTCTGAGCCGCGCTCTGGA GGCATTGGAACTCTGCAATTTATTGAGGAGTTTGTGCCATC GGTCTACTTTAACCCCTTCTCGGGACCTCCCGGCCACTATC CGGATCAATTTATTCCTAACTTTGACGCGGTAAAGGACTCG GCGGACGGCTACGACTGAATGTTAAGTGGAGAGGCAGAGC AACTGCGCCTGAAACACCTGGTCCACTGTCGCCGCCACAA GTGCTTTGCCCGCGACTCCGGTGAGTTTTGCTACTTTGAAT TGCCCGAGGATCATATCGAGGGCCCGGCGCACGGCGTCCG GCTTACCGCCCAGGGAGAGCTTGCCCGTAGCCTGATTCGG GAGTTTACCCAGCGCCCCCTGCTAGTTGAGCGGGACAGGG GACCCTGTGTTCTCACTGTGATTTGCAACTGTCCTAACCCT GGATTACATCAAGATCCTCTAGTTAATGTCAGGTCGCCTAA GTCGATTAACTAGAGTACCCGGGGATCTTATTCCCTTTAAC TAATAAAAAAAAATAATAAAGCATCACTTACTTAAAATCA GTTAGCAAATTTCTGTCCAGTTTATTCAGCAGCACCTCCTT GCCCTCCTCCCAGCTCTGGTATTGCAGCTTCCTCCTGGCTG CAAACTTTCTCCACAATCTAAATGGAATGTCAGTTTCCTCC TGTTCCTGTCCATCCGCACCCACTATCTTCATGTTGTTGCAG ATGAAGCGCGCAAGACCGTCTGAAGATACCTTCAACCCCG TGTATCCATATGACACGGAAACCGGTCCTCCAACTGTGCCT TTTCTTACTCCTCCCTTTGTATCCCCCAATGGGTTTCAAGAG AGTCCCCCTGGGGTACTCTCTTTGCGCCTATCCGAACCTCT AGTTACCTCCAATGGCATGCTTGCGCTCAAAATGGGCAAC GGCCTCTCTCTGGACGAGGCCGGCAACCTTACCTCCCAAA ATGTAACCACTGTGAGCCCACCTCTCAAAAAAACCAAGTC AAACATAAACCTGGAAATATCTGCACCCCTCACAGTTACCT CAGAAGCCCTAACTGTGGCTGCCGCCGCACCTCTAATGGTC GCGGGCAACACACTCACCATGCAATCACAGGCCCCGCTAA CCGTGCACGACTCCAAACTTAGCATTGCCACCCAAGGACC CCTCACAGTGTCAGAAGGAAAGCTAGCCCTGCAAACATCA GGCCCCCTCACCACCACCGATAGCAGTACCCTTACTATCAC TGCCTCACCCCCTCTAACTACTGCCACTGGTAGCTTGGGCA TTGACTTGAAAGAGCCCATTTATACACAAAATGGAAAACT AGGACTAAAGTACGGGGCTCCTTTGCATGTAACAGACGAC CTAAACACTTTGACCGTAGCAACTGGTCCAGGTGTGACTAT TAATAATACTTCCTTGCAAACTAAAGTTACTGGAGCCTTGG GTTTTGATTCACAAGGCAATATGCAACTTAATGTAGCAGG AGGACTAAGGATTGATTCTCAAAACAGACGCCTTATACTT GATGTTAGTTATCCGTTTGATGCTCAAAACCAACTAAATCT AAGACTAGGACAGGGCCCTCTTTTTATAAACTCAGCCCAC AACTTGGATATTAACTACAACAAAGGCCTTTACTTGTTTAC AGCTTCAAACAATTCCAAAAAGCTTGAGGTTAACCTAAGC ACTGCCAAGGGGTTGATGTTTGACGCTACAGCCATAGCCA TTAATGCAGGAGATGGGCTTGAATTTGGTTCACCTAATGCA CCAAACACAAATCCCCTCAAAACAAAAATTGGCCATGGCC TAGAATTTGATTCAAACAAGGCTATGGTTCCTAAACTAGG AACTGGCCTTAGTTTTGACAGCACAGGTGCCATTACAGTAG GAAACAAAAATAATGATAAGCTAACTTTGTGGACCACACC AGCTCCATCTCCTAACTGTAGACTAAATGCAGAGAAAGAT GCTAAACTCACTTTGGTCTTAACAAAATGTGGCAGTCAAAT ACTTGCTACAGTTTCAGTTTTGGCTGTTAAAGGCAGTTTGG CTCCAATATCTGGAACAGTTCAAAGTGCTCATCTTATTATA AGATTTGACGAAAATGGAGTGCTACTAAACAATTCCTTCCT GGACCCAGAATATTGGAACTTTAGAAATGGAGATCTTACT GAAGGCACAGCCTATACAAACGCTGTTGGATTTATGCCTA ACCTATCAGCTTATCCAAAATCTCACGGTAAAACTGCCAA AAGTAACATTGTCAGTCAAGTTTACTTAAACGGAGACAAA ACTAAACCTGTAACACTAACCATTACACTAAACGGTACAC AGGAAACAGGAGACACAACTCCAAGTGCATACTCTATGTC ATTTTCATGGGACTGGTCTGGCCACAACTACATTAATGAAA TATTTGCCACATCCTCTTACACTTTTTCATACATTGCCCAAG AATAAAGAATCGTTTGTGTTATGTTTCAACGTGTTTATTTTT CAATTGCAGAAAATTTCAAGTCATTTTTCATTCAGTAGTAT AGCCCCACCACCACATAGCTTATACAGATCACCGTACCTTA ATCAAACTCACAGAACCCTAGTATTCAACCTGCCACCTCCC TCCCAACACACAGAGTACACAGTCCTTTCTCCCCGGCTGGC CTTAAAAAGCATCATATCATGGGTAACAGACATATTCTTAG GTGTTATATTCCACACGGTTTCCTGTCGAGCCAAACGCTCA TCAGTGATATTAATAAACTCCCCGGGCAGCTCACTTAAGTT CATGTCGCTGTCCAGCTGCTGAGCCACAGGCTGCTGTCCAA CTTGCGGTTGCTTAACGGGCGGCGAAGGAGAAGTCCACGC CTACATGGGGGTAGAGTCATAATCGTGCATCAGGATAGGG CGGTGGTGCTGCAGCAGCGCGCGAATAAACTGCTGCCGCC GCCGCTCCGTCCTGCAGGAATACAACATGGCAGTGGTCTC CTCAGCGATGATTCGCACCGCCCGCAGCATAAGGCGCCTT GTCCTCCGGGCACAGCAGCGCACCCTGATCTCACTTAAATC AGCACAGTAACTGCAGCACAGCACCACAATATTGTTCAAA ATCCCACAGTGCAAGGCGCTGTATCCAAAGCTCATGGCGG GGACCACAGAACCCACGTGGCCATCATACCACAAGCGCAG GTAGATTAAGTGGCGACCCCTCATAAACACGCTGGACATA AACATTACCTCTTTTGGCATGTTGTAATTCACCACCTCCCG GTACCATATAAACCTCTGATTAAACATGGCGCCATCCACCA CCATCCTAAACCAGCTGGCCAAAACCTGCCCGCCGGCTAT ACACTGCAGGGAACCGGGACTGGAACAATGACAGTGGAG AGCCCAGGACTCGTAACCATGGATCATCATGCTCGTCATG ATATCAATGTTGGCACAACACAGGCACACGTGCATACACT TCCTCAGGATTACAAGCTCCTCCCGCGTTAGAACCATATCC CAGGGAACAACCCATTCCTGAATCAGCGTAAATCCCACAC TGCAGGGAAGACCTCGCACGTAACTCACGTTGTGCATTGTC AAAGTGTTACATTCGGGCAGCAGCGGATGATCCTCCAGTA TGGTAGCGCGGGTTTCTGTCTCAAAAGGAGGTAGACGATC CCTACTGTACGGAGTGCGCCGAGACAACCGAGATCGTGTT GGTCGTAGTGTCATGCCAAATGGAACGCCGGACGTAGTCA TATTTCCTGAAGCAAAACCAGGTGCGGGCGTGACAAACAG ATCTGCGTCTCCGGTCTCGCCGCTTAGATCGCTCTGTGTAG TAGTTGTAGTATATCCACTCTCTCAAAGCATCCAGGCGCCC CCTGGCTTCGGGTTCTATGTAAACTCCTTCATGCGCCGCTG CCCTGATAACATCCACCACCGCAGAATAAGCCACACCCAG CCAACCTACACATTCGTTCTGCGAGTCACACACGGGAGGA GCGGGAAGAGCTGGAAGAACCATGTTTTTTTTTTTATTCCA AAAGATTATCCAAAACCTCAAAATGAAGATCTATTAAGTG AACGCGCTCCCCTCCGGTGGCGTGGTCAAACTCTACAGCC AAAGAACAGATAATGGCATTTGTAAGATGTTGCACAATGG CTTCCAAAAGGCAAACGGCCCTCACGTCCAAGTGGACGTA AAGGCTAAACCCTTCAGGGTGAATCTCCTCTATAAACATTC CAGCACCTTCAACCATGCCCAAATAATTCTCATCTCGCCAC CTTCTCAATATATCTCTAAGCAAATCCCGAATATTAAGTCC GGCCATTGTAAAAATCTGCTCCAGAGCGCCCTCCACCTTCA GCCTCAAGCAGCGAATCATGATTGCAAAAATTCAGGTTCC TCACAGACCTGTATAAGATTCAAAAGCGGAACATTAACAA AAATACCGCGATCCCGTAGGTCCCTTCGCAGGGCCAGCTG AACATAATCGTGCAGGTCTGCACGGACCAGCGCGGCCACT TCCCCGCCAGGAACCATGACAAAAGAACCCACACTGATTA TGACACGCATACTCGGAGCTATGCTAACCAGCGTAGCCCC GATGTAAGCTTGTTGCATGGGCGGCGATATAAAATGCAAG GTGCTGCTCAAAAAATCAGGCAAAGCCTCGCGCAAAAAAG AAAGCACATCGTAGTCATGCTCATGCAGATAAAGGCAGGT AAGCTCCGGAACCACCACAGAAAAAGACACCATTTTTCTC TCAAACATGTCTGCGGGTTTCTGCATAAACACAAAATAAA ATAACAAAAAAACATTTAAACATTAGAAGCCTGTCTTACA ACAGGAAAAACAACCCTTATAAGCATAAGACGGACTACGG CCATGCCGGCGTGACCGTAAAAAAACTGGTCACCGTGATT AAAAAGCACCACCGACAGCTCCTCGGTCATGTCCGGAGTC ATAATGTAAGACTCGGTAAACACATCAGGTTGATTCACAT CGGTCAGTGCTAAAAAGCGACCGAAATAGCCCGGGGGAAT ACATACCCGCAGGCGTAGAGACAACATTACAGCCCCCATA GGAGGTATAACAAAATTAATAGGAGAGAAAAACACATAA ACACCTGAAAAACCCTCCTGCCTAGGCAAAATAGCACCCT CCCGCTCCAGAACAACATACAGCGCTTCCACAGCGGCAGC CATAACAGTCAGCCTTACCAGTAAAAAAGAAAACCTATTA AAAAAACACCACTCGACACGGCACCAGCTCAATCAGTCAC AGTGTAAAAAAGGGCCAAGTGCAGAGCGAGTATATATAGG ACTAAAAAATGACGTAACGGTTAAAGTCCACAAAAAACAC CCAGAAAACCGCACGCGAACCTACGCCCAGAAACGAAAG CCAAAAAACCCACAACTTCCTCAAATCGTCACTTCCGTTTT CCCACGTTACGTCACTTCCCATTTTAAGAAAACTACAATTC CCAACACATACAAGTTACTCCGCCCTAAAACCTACGTCACC CGCCCCGTTCCCACGCCCCGCGCCACGTCACAAACTCCACC CCCTCATTATCATATTGGCTTCAATCCAAAATAAGGTATAT TATTGATGAT 96 human IL-12 DNA GTCGACGCCACCATGTGTCACCAGCAGCTCGTGATTAGCT insert Sequence GGTTCAGCCTGGTGTTTCTGGCTAGCCCTCTGGTGGCCATC (including TGGGAGCTGAAGAAGGACGTGTACGTGGTGGAGCTCGACT restriction sites GGTACCCTGACGCTCCCGGCGAGATGGTCGTGCTGACCTG and Kozak CGACACCCCTGAGGAAGATGGCATCACCTGGACCCTGGAT sequence) CAAAGCTCCGAAGTGCTCGGCAGCGGCAAGACACTCACCA TCCAGGTGAAAGAGTTCGGAGACGCCGGCCAGTACACCTG CCACAAAGGCGGCGAGGTGCTGTCCCATTCCCTGCTGCTGC TGCACAAGAAAGAGGATGGCATCTGGTCCACCGACATCCT GAAGGACCAGAAGGAACCCAAGAACAAGACCTTTCTGAG ATGTGAGGCCAAGAACTACAGCGGCAGGTTCACCTGCTGG TGGCTGACAACAATCTCCACCGACCTGACCTTCAGCGTCAA GAGCAGCAGGGGCAGCAGCGACCCTCAAGGCGTGACATGT GGAGCCGCTACCCTGAGCGCTGAGAGAGTCAGGGGCGACA ATAAGGAGTACGAGTACTCCGTGGAATGCCAGGAGGACTC CGCCTGCCCTGCCGCCGAAGAGTCCCTCCCTATCGAAGTGA TGGTTGATGCCGTGCACAAGCTCAAGTATGAGAATTACAC CAGCAGCTTTTTCATCAGGGACATCATCAAGCCCGACCCCC CCAAAAACCTCCAGCTGAAACCCCTCAAGAATAGCAGGCA GGTGGAGGTCTCCTGGGAGTATCCTGACACCTGGAGCACC CCCCACAGCTACTTCTCCCTGACCTTCTGTGTGCAGGTGCA GGGCAAGAGCAAAAGGGAGAAGAAGGATAGGGTCTTTAC CGACAAGACCAGCGCCACAGTGATCTGCAGGAAGAACGCC AGCATTTCCGTCAGGGCCCAGGACAGGTACTACAGCAGCA GCTGGTCCGAGTGGGCTAGCGTGCCTTGTTCCGGCGGCGG AGGATCTGGCGGAGGCGGAAGTGGCGGAGGGGGCTCTAG AAACCTCCCCGTGGCCACACCCGACCCTGGCATGTTCCCCT GCCTCCACCACAGCCAGAACCTGCTGAGAGCCGTGAGCAA TATGCTGCAGAAGGCCAGGCAAACCCTGGAGTTCTACCCC TGTACCTCCGAGGAGATTGACCATGAGGACATCACAAAGG ACAAAACCAGCACCGTGGAGGCCTGTCTCCCCCTCGAACT GACCAAGAACGAGTCCTGCCTGAACTCCAGGGAGACATCC TTCATCACCAACGGCTCCTGCCTGGCCTCCAGAAAGACCA GCTTCATGATGGCCCTCTGCCTGAGCAGCATCTACGAGGAC CTCAAGATGTACCAGGTGGAGTTTAAAACAATGAACGCCA AGCTCCTCATGGACCCTAAGAGGCAGATTTTCCTCGACCAG AATATGCTGGCTGTCATTGACGAGCTGATGCAGGCCCTCA ATTTCAACTCCGAGACCGTCCCCCAGAAGTCCTCCCTGGAA GAGCCCGACTTTTACAAGACCAAGATCAAGCTCTGCATCCT GCTGCACGCCTTCAGAATTAGAGCCGTGACCATTGACAGG GTGATGAGCTACCTCAACGCCTCCTGATGACTCGAG 97 human IL-7 GTCGACGCCACCACATCCGCGGCAACGCCTCCTTGGTGTC insert GTCCGCTTCCAATAACCCAGCTTGCGTCCTGC ACACTTGTGGCTTCCGTGCACACATTAACAACTCATGGTTC TAGCTCCCAGTCGCCAAGC GTTGCCAAGGCGTTGAGAGATCATCTGGGAAGTCTTTTACC CAGAATTGCTTTGATTCAG GCCAGCTGGTTTTTCCTGCGGTGATTCGGAAATTCGCGAAT TCCTCTGGTCCTCATCCAG GTGCGCGGGAAGCAGGTGCCCAGGAGAGAGGGGATAATG AAGATTCCATGCTGATGATCC CAAAGATTGAACCTGCAGACCAAGCGCAAAGTAGAAACTG AAAGTACACTGCTGGCGGAT CCTACGGAAGTTATGGAAAAGGCAAAGCGCAGAGCCACGC CGTAGTGTGTGCCGCCCCCC TTGGGATGGATGAAACTGCAGTCGCGGCGTGGGTAAGAGG AACCAGCTGCAGAGATCACC CTGCCCAACACAGACTCGGCAACTCCGCGGAAGACCAGGG TCCTGGGAGTGACTATGGGC GGTGAGAGCTTGCTCCTGCTCCAGTTGCGGTCATCATGACT ACGCCCGCCTCCCGCAGAC CATGTTCCATGTTTCTTTTAGGTATATCTTTGGACTTCCTCC CCTGATCCTTGTTCTGTT GCCAGTAGCATCATCTGATTGTGATATTGAAGGTAAAGAT GGCAAACAATATGAGAGTGT TCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAA GAAATTGGTAGCAATTGCCT GAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATG CTAATAAGGAAGGTATGTT TTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAA TGAATAGCACTGGTGATTT TGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATAC TGTTGAACTGCACTGGCCA GGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAA CCAACAAAGAGTTTGGAAGA AAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTG TGTTTCCTAAAGAGACTATT ACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGGC ACTAAAGAACACTGAAAAAT ATGGAGTGGCAATATAGAAACACGAACTTTAGCTGCATCC TCCAAGAATCTATCTGCTTA TGCAGTTTTTCAGAGTGGAATGCTTCCTAGAAGTTACTGAA TGCACCATGGTCAAAACGG ATTAGGGCATTTGAGAAATGCATATTGTATTACTAGAAGAT GAATACAAACAATGGAAAC TGAATGCTCCAGTCAACAAACTATTTCTTATATATGTGAAC ATTTATCAATCAGTATAAT TCTGTACTGATTTTTGTAAGACAATCCATGTAAGGTATCAG TTGCAATAATACTTCTCAA ACCTGTTTAAATATTTCAAGACATTAAATCTATGAAGTATA TAATGGTTTCAAAGATTCA AAATTGACATTGCTTTACTGTCAAAATAATTTTATGGCTCA CTATGAATCTATTATACTG TATTAAGAGTGAAAATTGTCTTCTTCTGTGCTGGAGATGTT TTAGAGTTAACAATGATAT ATGGATAATGCCGGTGAGAATAAGAGAGTCATAAACCTTA AGTAAGCAACAGCATAACAA GGTCCAAGATACCTAAAAGAGATTTCAAGAGATTTAATTA ATCATGAATGTGTAACACAG TGCCTTCAATAAATGGTATAGCAAATGTTTTGACATGAAAA AAGGACAATTTCAAAAAAA TAAAATAAAATAAAAATAAATTCACCTAGTCTAAGGATGC TAAACCTTAGTACTGAGTTA CATTGTCATTTATATAGATTATAACTTGTCTAAATAAGTTT GCAATTTGGGAGATATATT TTTAAGATAATAATATATGTTTACCTTTTAATTAATGAAAT ATCTGTATTTAATTTTGAC ACTATATCTGTATATAAAATATTTTCATACAGCATTACAAA TTGCTTACTTTGGAATACA TTTCTCCTTTGATAAAATAAATGAGCTATGTATTAAAAAAA AAAAAAAA 98 human CD70 NCBI GTCGACGCCACCCCAGAGAGGGGCAGGCTGGTCCCCTGA insert Reference CAGGTTGAAGCAAGTAGACGCCCAGGAGCCCCG Sequence: GGAGGGGGCTGCAGTTTCCTTCCTTCCTTCTCGGCAGCGCT NM_001252.4 CCGCGCCCCCATCGCCCCT CCTGCGCTAGCGGAGGTGATCGCCGCGGCGATGCCGGAGG AGGGTTCGGGCTGCTCGGTG CGGCGCAGGCCCTATGGGTGCGTCCTGCGGGCTGCTTTGGT CCCATTGGTCGCGGGCTTG GTGATCTGCCTCGTGGTGTGCATCCAGCGCTTCGCACAGGC TCAGCAGCAGCTGCCGCTC GAGTCACTTGGGTGGGACGTAGCTGAGCTGCAGCTGAATC ACACAGGACCTCAGCAGGAC CCCAGGCTATACTGGCAGGGGGGCCCAGCACTGGGCCGCT CCTTCCTGCATGGACCAGAG CTGGACAAGGGGCAGCTACGTATCCATCGTGATGGCATCT ACATGGTACACATCCAGGTG ACGCTGGCCATCTGCTCCTCCACGACGGCCTCCAGGCACCA CCCCACCACCCTGGCCGTG GGAATCTGCTCTCCCGCCTCCCGTAGCATCAGCCTGCTGCG TCTCAGCTTCCACCAAGGT TGTACCATTGCCTCCCAGCGCCTGACGCCCCTGGCCCGAGG GGACACACTCTGCACCAAC CTCACTGGGACACTTTTGCCTTCCCGAAACACTGATGAGAC CTTCTTTGGAGTGCAGTGG GTGCGCCCCTGACCACTGCTGCTGATTAGGGTTTTTTAAAT TTTATTTTATTTTATTTAA GTTCAAGAGAAAAAGTGTACACACAGGGGCCACCCGGGGT TGGGGTGGGAGTGTGGTGGG GGGTAGTGGTGGCAGGACAAGAGAAGGCATTGAGCTTTTT CTTTCATTTTCCTATTAAAA AATACAAAAATCA 99 NV1 Adenovirus GGCGGAAGTGTGATGTTGCAAGTGTGGCGGAACACATGTA E1a AGCGACGGATGTGGCGCAAGTCTATGTTGTAGTAAATTTG Enhancer GGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAAC Region TGAATAAGAGGAAGTGAAATC Mutant 1 100 NV2 Adenovirus ACAGGAAGTGACATGTTGCAAGTGTGGCGGAACACATGTA E1a AGCGACGGATGTGGCGCAAGTCTATGTTGTAGTAAATTTG Enhancer GGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAAC Region TGAATAAGAGGAAGTGAAATCT Mutant 2 101 NV3 Adenovirus ACAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTG E1a GCGCAAGTCTATGTTGTAGTAAATTTGGGCGTAACCGAGT Enhancer AAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGAGGAA Region GTGAAATCT Mutant 3 102 NV4 Adenovirus GGCGGAAGTGTGATGTTGCAAGTGTGGCGGAACACATGTA E1a AGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCC Enhancer GGTGTACGGCGGAAGTGTGAATTTTCGCGCGGTTTTAGAC Region GGATGTGGCAGTAAATTTGGGCGTAACCGAGTAAGATTTG Mutant 4 GCCATTTTCGCGGGAAAACTGAATAAGAGGAAGTGAAATC T 103 NV5 Adenovirus GGCGGAAGTGTGATGTTGCAAGTGTGGCGGAACACATGTA E1a AGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCC Enhancer GGTGTACGGCGGAAGTGTGAATTTTCGCGCGGTTTTAGAC Region GGATGTGGCAGTAAATTTGGGCGTAACCGAGTAAGATTTG Mutant 5 GCCATTTTCGCGGGAAAACTGAATAAGAGGATGTGAAATC T 104 NV6 Adenovirus GGCGGAAGTGTGATGTTGCAAGTGTGGCGGAACACATGTA E1a AGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCC Enhancer GGTGTACGGCGGAAGTGTGAATTTTCGCGCGGTTTTAGAC Region GGATGTGGCAGTAAATTTGGGCGTAACCGAGTAAGATTTG Mutant 6 GCCATTTTCGCGGGAAAACTGAATAGGCGGAAGTGTGATC T 105 NV7 Adenovirus GGCGGAAGTGTGATGTTGCAAGTGTGGCGGAACACATGTA E1a AGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCC Enhancer GGTGTACACAGGAAGTGACAATTTTCGCGCGGTTTTAGAC Region GGATGTGGCAGTAAATTTGGGCGTAACCGAGTAAGATTTG Mutant 7 GCCATTTTCGCGGGAAAACTGAATAGGCGGAAGTGTGATC T 

What is claimed is:
 1. A pharmaceutical composition comprising an effective amount of a recombinant adenoviral vector comprising: a transgene insertion site located between the start site of adenoviral E1b-19K and the start site of adenoviral E1b-55K, wherein a first DNA sequence and a second DNA sequence are each inserted into the transgene insertion site; wherein the first DNA sequence encodes a polypeptide selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, and a human OX40 ligand, and wherein the second DNA sequence encodes a polypeptide selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, and a human OX40 ligand; wherein the adenoviral vector comprises a modified adenoviral E1a regulatory sequence wherein at least one Pea3 binding site, or a functional portion thereof, of the recombinant adenoviral vector is modified or deleted.
 2. The pharmaceutical composition of claim 1, wherein the adenoviral vector comprises an IRES element or encodes a self-cleaving 2A peptide sequence between the first DNA sequence and the second DNA sequence.
 3. The pharmaceutical composition of claim 1, comprising a modified E3 region.
 4. The pharmaceutical composition of claim 1, comprising an intact E3 region.
 5. The pharmaceutical composition of claim 3, further comprising a third DNA sequence inserted into the E3 region, wherein the third DNA sequence encodes a polypeptide selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, or a human OX40 ligand.
 6. The pharmaceutical composition of claim 1, wherein the adenoviral vector comprises a nucleic acid sequence at least 95% identical in an E3 region to vector d1327.
 7. The pharmaceutical composition of claim 1, wherein the chimeric human IL-12 polypeptide comprises a p40 polypeptide, a p35 polypeptide, and a linker polypeptide.
 8. The pharmaceutical composition of claim 1, wherein the chimeric human IL-12 polypeptide comprises a sequence as set forth in SEQ ID NO:46.
 9. The pharmaceutical composition of claim 1, formulated for systemic administration.
 10. The pharmaceutical composition of claim 1, formulated for intratumoral administration.
 11. A pharmaceutical composition comprising an effective amount of a recombinant adenoviral vector comprising: a. a first transgene insertion site located between the start site of adenoviral E1b-19K and the start site of adenoviral E1b-55K; b. a second transgene insertion site located in adenoviral E3 region; c. a first DNA sequence, present in the first transgene insertion site, encoding one or a plurality of polypeptides selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, and a human OX40 ligand; and d. a second DNA sequence, present in the second transgene insertion site, encoding one or a plurality of polypeptides selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, and a human OX40 ligand, wherein the adenoviral vector comprises a modified adenoviral E1a regulatory sequence.
 12. The pharmaceutical composition of claim 11, further comprising a third DNA sequence inserted into a third transgene insertion site, encoding one or a plurality of polypeptides selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, and a human OX40 ligand.
 13. The pharmaceutical composition of claim 8, wherein at least one of the first DNA sequence, the second DNA sequence, and the third DNA sequence independently comprises an IRES element and/or a self-cleaving 2A peptide.
 14. The pharmaceutical composition of claim 11, wherein at least one E1a regulatory sequence Pea3 binding site, or a functional portion thereof of the adenoviral vector, is modified or deleted.
 15. The pharmaceutical composition of claim 11, wherein a sequence between two Pea3 sites of the adenoviral vector is deleted.
 16. The pharmaceutical composition of claim 11, comprising a modified E3 region.
 17. The pharmaceutical composition of claim 11, wherein the adenoviral vector comprises a nucleic acid sequence at least 95% identical in an E3 region to vector d1327.
 18. The pharmaceutical composition of claim 11, wherein the chimeric human IL-12 polypeptide comprises a p40 polypeptide, a p35 polypeptide, and a linker polypeptide.
 19. A method for treating a tumor in a human subject in need thereof, comprising administering to the human with the tumor a therapeutic amount of the pharmaceutical composition of claim 1 by systemic or intratumoral administration.
 20. A method for treating a tumor in a human subject in need thereof, comprising administering to the human with the tumor a therapeutic amount of the pharmaceutical composition of claim 11 by systemic or intratumoral administration. 